Long-term outcome study of growth factor-treated pressure ulcers.

BACKGROUND: Exogenous application of growth factors have been reported in an attempt to accelerate healing of chronic wounds. Most of the trials were of brief duration with short to no follow-up periods. Long-term outcome studies are sparse for pressure ulcer therapies with success rates around 30% for both operative and nonoperative treatments. METHODS: Follow-up evaluations were performed serially up to 12 months for patients completing a 35 day blinded, placebo-controlled cytokine clinical trial of pressure ulcers. RESULTS: Fifty-four of 61 patients completed the follow-up period with 68.5% of the patients (37 of 54) being healed after 1 year. Of patients healing > or =85% during the active treatment phase, 84.6% were healed after 1 year compared with 61% of those that healed <85% during treatment (P <0.05). CONCLUSION: Long-term outcome was better in this growth factor trial than with surgical or standard nonoperative treatment of pressure ulcers. Since only patients receiving exogenously applied cytokines achieved >85% closure during the treatment phase of the trial, the excellent long-term outcome appears attributable to the cytokine therapy.

Sequential cytokine therapy for pressure ulcers: clinical and mechanistic response.

OBJECTIVE: To compare the healing response of sequential topically applied cytokines to that of each cytokine alone and to a placebo in pressure ulcers, and to evaluate the molecular and cellular responses. SUMMARY BACKGROUND DATA: Because of a deficiency of cytokine growth factors in chronic wounds and the reversal of impaired healing in animal models, pressure ulcer trials have been performed with several exogenously applied growth factors. Because single-factor therapy has not been uniformly successful, combination or sequential cytokine therapy has been proposed. Laboratory data have suggested that sequential treatment with granulocyte-macrophage/colony-stimulating factor (GM-CSF)/basic fibroblast growth factor (bFGF) might augment the previously reported effect of bFGF alone. METHODS: A masked, randomized pressure ulcer trial was performed comparing sequential GM-CSF/bFGF therapy with that of each cytokine alone and with placebo during a 35-day period. The primary measure was wound volume decrease over time. Cytokine wound levels and mRNA levels were serially determined. Fibroblast-populated collagen lattices (FPCLs) were constructed from serial fibroblast biopsies. Cellular ultrastructure was evaluated by electron microscopy. Changes in ease of surgical closure and its relative cost were determined. RESULTS: Ulcers treated with cytokines had greater closure than those in placebo-treated patients. Patients treated with bFGF alone did the best, followed by the GM-CSF/bFGF group. Patients treated with GM-CSF or bFGF had higher levels of their respective cytokine after treatment. Patients with the greatest amount of healing showed higher levels of platelet-derived growth factor (PDGF) on day 10 and transforming growth factor beta (TGFbeta1) on day 36. Message for the bFGF gene was upregulated after treatment with exogenous bFGF, suggesting autoinduction of the cytokine. FPCLs did not mimic the wound responses. Ultrastructure of wound biopsies showed response to bFGF. Treatment with any of the cytokines improved the wound by allowing easier wound closure. This was most marked for the bFGF-alone treatment, with a cost savings of $9,000 to $9,200. CONCLUSIONS: Treatment with bFGF resulted in significantly greater healing than the other treatments in this trial. The clinical response appeared to be related to upregulation of the bFGF message and to increased levels of PDGF-AB, bFGF, and TGFbeta1 in the wounds and changes in ultrastructure. The resultant improvements could be correlated with cost savings.

Safety and effect of transforming growth factor-beta(2) for treatment of venous stasis ulcers.

Transforming growth factor-beta(2) promotes healing in a variety of animal models and exhibits clinical effects thought to be mediated by connective tissue formation. Two clinical trials were conducted to evaluate the safety and effect of transforming growth factor-beta(2) purified from bovine bone and delivered topically to venous stasis ulcers three times per week for up to 6 weeks by means of a lyophilized collagen vehicle. The first was an open-label trial comparing transforming growth factor-beta(2) purified from bovine bone (0.5 microg/cm(2)) with a placebo consisting of lyophilized collagen vehicle-without active drug. After no safety issues arose in that trial, a prospectively randomized, closed-label, observer-blinded, three-armed trial was conducted to compare bovine transforming growth factor-beta(2) (2.5 microg/cm(2)) with the collagen matrix placebo vehicle and with a standard dressing. Standardized elastic compression was applied to all test extremities. The rate of reduction of ulcer area as measured by planimetry was the primary measure of effect. No serious safety-related events occurred in either trial. Clinical evaluation suggested that improvement in the quality and quantity of granulation tissue appeared to precede epithelialization of ulcers treated with bovine transforming growth factor-beta(2). In both studies, treatment with bovine transforming growth factor-beta(2) appeared to have a positive effect on the rate of ulcer closure, whereas ulcers in the control groups continued to exhibit impaired healing. In the open-label study, the mean rate of closure of ulcers treated with bovine transforming growth factor-beta(2) was significantly greater than that of ulcers treated with placebo. There was likewise enhanced reduction in ulcer area in the ulcers treated with bovine transforming growth factor-beta(2) in the second trial. However, because of a higher variability in patient response and a greater placebo effect, the difference was not significant. The placebo was not worse than the standard care arm, thereby showing that the vehicle is not injurious to healing. The combined results of the two trials suggest that, at doses of 0.5 to 2.5 microg/cm(2), bovine transforming growth factor-beta(2) is safe as a topically applied agent in a collagen matrix vehicle and can have a positive effect on closure of venous stasis ulcers. Large multicenter trials appear to be indicated to evaluate fully the potential utility of transforming growth factor-beta(2) in accelerating closure of chronic dermal ulcers.

Safety and effect of topical recombinant human interleukin-1beta in the management of pressure sores.

Interleukin-1beta is produced by numerous cell types including monocytes and fibroblasts. It has been shown to stimulate multiple cell types including fibroblasts, keratinocytes, endothelial cells, neutrophils, macrophages, and lymphocytes. Previously, interleukin-1beta was shown to accelerate healing in partial-thickness and full-thickness wounds in animals and was also shown to be safe when applied topically in Phase I human trials. Therefore a prospectively randomized, blind, placebo-controlled trial was performed with patients with chronic pressure ulcers. Doses of interleukin-1beta of .01 microg, .10 microg, and 1.0 microg per square centimeter did not show acceleration of healing of the pressure ulcers. Therefore use of recombinant human interleukin-1beta in this study was safe but, at the dose levels tested, did not result in improvement in the healing ratio.

Recombinant human platelet-derived growth factor-BB for the treatment of chronic pressure ulcers.

A randomized phase I/II double-blind, placebo-controlled study was designed to evaluate 1, 10, and 100 micrograms/ml (0.01, 0.1, and 1.0 micrograms/cm2) recombinant human BB homodimeric platelet-derived growth factor (rPDGF-BB) applied topically to chronic pressure ulcers for 28 days. Twenty patients were enrolled and completed the trial. No toxicities were associated with rPDGF-BB treatment. Patients treated with 100 micrograms/ml of rPDGF-BB had a pronounced healing response compared with placebo-treated patients. By day 29, ulcers treated with 100 micrograms/ml of rPDGF-BB were smaller in remaining size compared with those of placebo-treated patients when the following specific parameters were measured: percentage of initial depth (14.1 +/- 7.4 vs. 34.9 +/- 6.7) and percentage of initial volume (6.4 +/- 4.0 vs. 21.8 +/- 5.6). Histological analyses of biopsies revealed active wound healing processes in all groups with no disruption in the normal healing sequence in rPDGF-BB-treated wounds. The results of this small, descriptive study suggest rPDGF-BB is a potent vulnerary agent for accelerating soft-tissue repair, warranting further study.

Platelet-derived growth factor BB for the treatment of chronic pressure ulcers.

A randomised, phase I/II, double-blind, placebo-controlled study was designed to assess the effect of topically applied recombinant human BB homodimeric platelet-derived growth factor (rPDGF-BB) on healing of chronic pressure ulcers. Twenty patients were randomly allocated daily treatment for 28 days with 1, 10, or 100 micrograms/ml rPDGF-BB (0.01, 0.1, or 1.0 micrograms per cm2 ulcer area) or placebo. Patients treated with 100 micrograms/ml rPDGF-BB showed a greater healing response than the placebo group, but the lower doses had little effect. After 28 days, ulcers treated with 100 micrograms/ml rPDGF-BB were smaller than those treated with placebo (mean [SE] volume 6.4 [4.0] vs 21.8 [5.6]% of day 0 volume). There were no toxic effects. These preliminary findings suggest that rPDGF-BB is a potent wound-healing agent in soft tissue.

Effect of Tris, HEPES, and TES buffers on binding at mu-, delta-, and kappa-opioid sites in guinea pig brain.

In homogenates of guinea-pig brain minus cerebellum, the delta-binding of 1.5 nM [3H]-[D-Pen2,D-Pen5]enkephalin is little affected by the type and concentration of the three buffers, Tris-HCl, HEPES-KOH, or TES-KOH (10-75 mM). However, the mu-binding of 1 nM [3H]-[D]Ala2,MePhe4,Gly-ol5]enkephalin or the kappa-binding of 1.5 nM [3H]-U-69,593 is influenced by the choice of buffer. A suitable concentration of buffer for further analyses of opioid binding has been found to be 10 mM. At each site, the effects of MgCl2 on binding are the same whether 10 mM Tris-HCl, HEPES-KOH, or TES-KOH are used but variations of the effects of NaCl confirm the view that mu- and kappa-sites, but not delta-sites, are affected by choice of buffer. Furthermore, under some assay conditions the effects of NaCl and MgCl2 at the kappa-sites of guinea-pig cerebellum differ from their effects in brain minus cerebellum, indicating that these are differences of binding characteristics at the kappa-sites of these tissues.

Regional variations in binding capacities at mu-, delta- and kappa-opioid sites in membrane suspensions from rabbit brain.

The highest maximum binding capacities at the mu-sites of rabbit brain are in the striatum, with intermediate levels in the diencephalon, mesencephalon, cerebellum and cortex and low levels in the pons-medulla and hippocampus. For the delta-site the highest maximum binding capacity is also in the striatum; there are almost equally low levels in the other brain regions. At the kappa-sites the maximum binding capacities are highest in the diencephalon; there are intermediate levels in the cortex and striatum, and low levels in the mesencephalon, cerebellum, hippocampus and pons-medulla. The KD values lack reproducibility; there are no regional variations at the kappa-site as estimated with [3H](-)-bremazocine, but the possibility cannot be excluded that there are regional variations in the KD values for [3H][D-Ala2,MePhe4,Gly-ol5]enkephalin at the mu-site or for [3H][D-Ala2,D-Leu5]enkephalin at the delta-site. It may be important to use saturation analysis in future investigations of the distributions of the binding sites.

Modulation of binding at opioid receptors by mono- and divalent cations and by cholinergic compounds.

In membrane suspensions from guinea-pig brain, NaCl, LiCl, NH4Cl and KCl, inhibit the equilibrium binding (25 degrees C) of the selective mu-agonist [3H]-[D-Ala2,MePhe4,Gly-ol5]enkephalin, the selective delta-agonist [3H]-[D-Pen2,D-Pen5]enkephalin and the selective kappa-agonist [3H]-dynorphin A (1-9). Choline chloride inhibits the binding of the mu- and kappa-agonists but not of the delta-agonist; the choline derivative, methacholine, inhibits also the binding of the delta-agonist. Binding of the delta-agonist is potentiated by CaCl2, MgCl2 and MnCl2; these salts inhibit binding of the kappa-agonist. As far as binding of the mu-agonist is concerned, MgCl2 and MnCl2 may potentiate or inhibit whereas CaCl2 is only inhibitory. The binding of the mu-antagonist [3H]-naloxone is potentiated by NaCl; while the threshold of inhibition by LiCl is increased there is no potentiation. In membrane suspensions of the rabbit cerebellum about 80% of the opioid binding sites are of the mu-type; the binding of the mu-agonist [3H]-[D-Ala2,MePhe4,Gly-ol5]enkephalin is inhibited by NaCl, LiCl, KCl and choline chloride whereas that of the mu-antagonists [3H]-naloxone and [3H]-(-)-bremazocine is potentiated at low concentrations but inhibited at higher concentrations of NaCl. In membranes of the guinea-pig cerebellum about 80% of the opioid binding sites are of the kappa-type; they are particularly effective for assays of kappa-receptors when the selective kappa-agonist [3H]-dynorphin A (1-9) is used as ligand.

Effects of cations on binding, in membrane suspensions, of various opioids at mu-sites of rabbit cerebellum and kappa-sites of guinea-pig cerebellum.

At the mu-sites of rabbit cerebellum, NaCl, LiCl, KCl, choline chloride and MnCl2 were tested for potentiation and inhibition of the binding of several opioids. Naloxone, (-)-bremazocine and diprenorphine are mu-antagonists in pharmacological assays and their binding is potentiated by the lower concentrations and inhibited by the higher concentrations of NaCl. The binding of the agonists [3H]-[D-Ala2, MePhe4, Gly-ol5]enkephalin and [3H]-dihydromorphine is inhibited. MnCl2 potentiates the binding of the agonist [3H]-[D-Ala2, MePhe4, Gly-ol5]enkephalin but not the binding of the antagonists. The thresholds of inhibition and slopes of the dose-response curves for inhibition by MnCl2 and LiCl vary. This finding may indicate that potentiating effects of MnCl2 and LiCl are masked by simultaneous inhibition. At the kappa-sites of guinea-pig cerebellum, NaCl, KCl and MnCl2 inhibit the binding of [3H]-dynorphin A (1-8), [3H]-dynorphin A (1-9), [3H]-(-)-bremazocine, [3H]-tifluadom, and [3H]-diprenorphine. NaCl also causes a small potentiation of the binding of [3H]-diprenorphine, which is a kappa-agonist in the guinea-pig myenteric plexus but a kappa-antagonist in the rabbit vas deferens. The slopes of the inhibitory dose-response curves and the thresholds of inhibition vary with the different ligands. Therefore some potentiating effects may have been masked. The results support the view that NaCl, and perhaps LiCl, but not KCl and choline chloride, potentiate the binding of mu-antagonists but not the binding of mu-agonists. It is not yet possible to decide whether, at the kappa-site, there is a similar differentiation of the binding of agonists and antagonists.

Differential postnatal development of mu-, delta- and kappa-opioid binding sites in rat brain.

With selective labelling techniques and analysis of saturation curves it is shown that in rat brain the concentrations of mu-, delta- and kappa-binding sites increase differentially during postnatal development. There are no changes in the binding affinities. The concentration (pmol/g brain) of kappa-sites are first to reach adult levels, namely between 7 and 14 days after birth. Adult levels of mu-sites are attained between 14 and 21 days after birth. The most striking finding is that development of delta-sites, which are not detectable 3 days after birth by the method used, lags markedly behind that of mu- and kappa-sites. The profile of development in rat brain is compared to that found previously in mouse brain.

Control by cations of opioid binding in guinea pig brain membranes.

In membrane suspensions from guinea pig brain or cerebellum, NaCl, LiCl, NH4Cl, and KCl inhibit the equilibrium binding at 25 degrees C of the selective mu-agonist [3H][2-D-alanine,4-methylphenylalanine,5-glycinol]enkephalin ([D-Ala2,MePhe4,Gly-ol5]EK), the selective delta-agonist [3H][2-D-penicillamine,5-D-penicillamine]enkephalin ([D-Pen2,D-Pen5]-EK), and the selective kappa-agonist [3H]dynorphin A-(1-9). Choline chloride inhibits mu- and kappa-binding but not delta-binding. The relative activities of these monovalent salts and the slopes of the dose-response curves are site-dependent. Binding at the kappa-binding site is also inhibited by CaCl2, MnCl2, and MgCl2. On the other hand, these divalent salts potentiate delta-binding, and MnCl2 and MgCl2 have both potentiating and inhibitory effects on mu-binding; CaCl2 inhibits but does not potentiate mu-binding. Thus, the mechanisms by which monovalent cations inhibit opioid binding differ from those of divalent cations, and the mechanisms of action of both monovalent and divalent cations may differ at each site. When the antagonist [3H]naloxone, rather than the agonist [3H][D-Ala2,MePhe4,Gly-ol5]EK, is used to label the mu-binding site, the main effect of NaCl is to potentiate binding; a 22-fold higher concentration of LiCl is required to inhibit binding. The effects of NH4Cl, KCl, MnCl2, MgCl2, CaCl2, and choline chloride are little changed when [3H]naloxone is the ligand.

Site-dependent effects of ions on mu-, delta- and kappa-opioid binding in suspensions of guinea-pig brain membranes.

NaCl, LiCl, NH4Cl and KCl inhibit the equilibrium binding of peptide agonists at each of the mu-, delta- and kappa-sites; the orders of potencies and the slopes of the dose-response curves are site-dependent. In contrast, the monovalent salt choline chloride inhibits mu- and kappa-binding but has little effect on delta-binding. The profiles of activity of MnCl2, CaCl2 and MgCl2 are also site-dependent but differ markedly from those of the monovalent salts.

Differential postnatal development of mu-, delta-and chi-opioid binding sites in mouse brain.

By selective labelling techniques together with analysis of saturation curves it is shown that the concentrations of the mu-, delta- and chi-binding sites increase during postnatal development particularly in the first few weeks after birth. There are little or no changes in affinity. The rate of development is different for each type of site. The most striking finding is that the development of mu- and chi-sites precedes that of delta-sites.

Kappa-binding and degradation of [3H]dynorphin A (1-8) and [3H]dynorphin A (1-9) in suspensions of guinea pig brain membranes.

Following incubation of [3H]dynorphin A (1-8) and [3H]dynorphin A (1-9) with suspensions of guinea pig brain membranes, analysis of the supernatants by HPLC has shown that both peptides are degraded at 25 degrees C and at 0 degrees C. Bestatin and captopril reduce degradation at 0 degrees C but for a similar degree of protection at 25 degrees C arginine-containing dipeptides are also required. The effects of these peptidase inhibitors on the degradation profiles indicate that [3H]dynorphin A (1-8) has three main sites of cleavage: the Tyr1-Gly2, Arg6-Arg7, and Leu5-Arg6 bonds. With [3H]dynorphin A (1-9) as substrate the Arg7-Ile8 and Ile8-Arg9 bonds are also liable to cleavage. In binding assays, in contrast to the effects of peptidase inhibitors on the degradation of unbound [3H]dynorphin A (1-8) and [3H]dynorphin A (1-9), bestatin and captopril have little effect on the binding characteristics of the tritiated dynorphin A fragments at the kappa-site at 0 degrees C. However, at 25 degrees C binding is low in the absence of peptidase inhibitors. When binding at mu- and delta-sites is prevented, the maximal binding capacities of [3H]dynorphin A (1-8), [3H]dynorphin A (1-9), and [3H](-)-bremazocine at the kappa-site are similar; [3H]dynorphin A (1-9) has 5-10 times higher affinity for the kappa-site than [3H]dynorphin A (1-8). Comparison of the effects of peptidase inhibitors on unbound dynorphin A fragments with their effects in binding assays suggests that the bound peptides are protected from the action of peptidases.

Pre-incubation of guinea-pig myenteric plexus with beta-funaltrexamine: discrepancy between binding assays and bioassays.

The acute effects of beta-funaltrexamine and the effects of pre-incubation with this compound were examined in five in vitro assay tissues and in selective binding assays in homogenates of guinea-pig brain and myenteric plexus. In competitive displacement assays with selective ligands, beta-funaltrexamine had highest affinity for the mu-binding site in the myenteric plexus and brain of guinea-pig. Its affinity for the kappa-site was about 15% of that for the mu-site. Pre-incubation of the assay tissues with beta-funaltrexamine caused an increase in the IC50 values of mu- and delta-receptor agonists but not of kappa-agonists. Although in bioassays on the myenteric plexus-longitudinal muscle preparation of the guinea-pig, the IC50 value of the mu-receptor ligand [D-Ala2, MePhe4, Gly-ol5] enkephalin was increased up to 124 fold, its binding at the mu-site in homogenates of the preparation was not affected by this treatment. These findings indicate that the effects of pre-incubation with beta-funaltrexamine on agonist potency of the mu-receptor ligand are due to an interference with the coupling mechanism between the mu-binding site and the effector system.

Species differences in the concentrations and distributions of opioid binding sites.

Binding at the mu, delta- and kappa-types of opioid binding sites was compared in homogenates from the brains of guinea-pig, rabbit, rat and two mouse strains, under conditions of selective labelling. Species differences were shown by two observations. Firstly, analysis of saturation curves in homogenates of brain from which the cerebellum had been removed showed that in guinea-pig brain the opioid binding sites consist of 24% mu-sites, 32% delta-sites and 44% kappa-sites. In contrast, in rabbit brain the corresponding values are 43% mu-sites, 19% delta-sites and 37% kappa-sites and in rat brain, 46% mu-sites, 42% delta-sites and 12% kappa-sites. In the brains of DBA/2 mice the opioid binding sites are comprised of 51% mu-sites, 29% delta-sites and 20% kappa-sites and in C57BL/10 mice, of 44% mu-sites, 35% delta-sites and 21% kappa-sites; these strain differences are due to significant differences in the concentrations of the mu-sites. Secondly, species differences were found when the binding of single concentrations of tritiated ligands (1 X KD value in whole brain) was determined at mu-, delta- and kappa-sites in six brain regions from guinea-pig, rat or rabbit.

[3H]-Dynorphin A (1-8): degradation profile and binding characteristics in brain homogenates.

[3H]-Dynorphin A (1-8) was degraded in brain homogenates at 25 degrees and even at 0 degree C. The peptidase inhibitors bestatin and captopril almost completely protected[3H]-dynorphin A (1-8) from degradation at 0 degree C but had only little effect on binding at this temperature. At 25 degrees C, the binding of [3H]-dynorphin A (1-8) was markedly improved by addition of bestatin, captopril and L-leucyl-L-arginine, which afforded some, but not complete protection from degradation. The results of saturation binding assays at 25 degrees C in the presence of the peptidase inhibitors were variable. However, it was found from saturation binding assays at 0 degree C that the maximum binding capacity for [3H]-dynorphin A (1-8) at the kappa-site is similar to that of [3H]-(-)-bremazocine and [3H]-dynorphin A (1-9).