Antibacterial bioagents based on principles of bacteriophage biology: an overview.

Bacteriophages were discovered almost a century ago. With the advent of antibiotics, the use of bacteriophages for treatment of infections fell out of favor in Western medicine. In light of the rise of antibiotic resistance, phages and their products (lysins) are rediscovered as antibacterial bioagents. This overview summarizes principles of phage biology and their translation for therapeutic and preventive applications. Examples are presented to highlight their therapeutic promise for prophylaxis and treatment of bacterial infections including multidrug-resistant organisms in humans and animals, and their use as decontaminants of food supplies and environments. Besides research on the in vivo behavior of phages and lysins, dialogues between researchers and regulatory agencies are necessary to publish guidelines for bacteriophage manufacturing and formulation for human use. Only well-designed, double-blind randomized controlled trials will determine if phages and lysins are safe and effective adjuncts or alternatives to antibiotic therapy for infections with multidrug-resistant organisms.

Safety Studies of Pneumococcal Endolysins Cpl-1 and Pal.

Bacteriophage-derived endolysins have gained increasing attention as potent antimicrobial agents and numerous publications document the in vivo efficacy of these enzymes in various rodent models. However, little has been documented about their safety and toxicity profiles. Here, we present preclinical safety and toxicity data for two pneumococcal endolysins, Pal and Cpl-1. Microarray, and gene profiling was performed on human macrophages and pharyngeal cells exposed to 0.5 µM of each endolysin for six hours and no change in gene expression was noted. Likewise, in mice injected with 15 mg/kg of each endolysin, no physical or behavioral changes were noted, pro-inflammatory cytokine levels remained constant, and there were no significant changes in the fecal microbiome. Neither endolysin caused complement activation via the classic pathway, the alternative pathway, or the mannose-binding lectin pathway. In cellular response assays, IgG levels in mice exposed to Pal or Cpl-1 gradually increased for the first 30 days post exposure, but IgE levels never rose above baseline, suggesting that hypersensitivity or allergic reaction is unlikely. Collectively, the safety and toxicity profiles of Pal and Cpl-1 support further preclinical studies.

Targeted anti-staphylococcal therapy with endolysins in atopic dermatitis and the effect on steroid use, disease severity and the microbiome: study protocol for a randomized controlled trial (MAAS trial).

BACKGROUND: Atopic dermatitis (AD) is associated with reduced skin microbial diversity and overgrowth of Staphylococcus (S.) aureus. However, the importance of S. aureus colonisation in the complex pathogenesis remains unclear and studies on the effect of anti-staphylococcal therapy in non-infected AD show contradictory results. Long-term interventions against S. aureus might be needed to restore the microbial balance, but carry the risk of bacterial resistance induction. Staphefekt, an engineered bacteriophage endolysin, specifically kills S. aureus leaving other skin commensals unharmed. Bacterial resistance towards endolysins has not been reported, nor is it expected, which allows us to study its effect as long-term anti-staphylococcal treatment in non-infected AD. METHODS: This is a multi-centre, placebo-controlled, double-blinded and randomized superiority trial with a parallel group design. A total of 100 participants, aged 18 years or older, diagnosed with moderate to severe AD and using a topical corticosteroid in the weeks before enrolment are included in the study. The study is executed in the Erasmus MC University Medical Centre Rotterdam in collaboration with the Havenziekenhuis Rotterdam. After a 2-week run-in period to standardize the corticosteroid use with triamcinolone acetonide 0.1% cream, participants will be randomized to either treatment with Staphefekt in a cetomacrogol-based cream or a placebo for 12 weeks, followed by an 8-week follow-up period. The primary objective is to assess the difference in the need for corticosteroid co-therapy between the Staphefekt and the placebo group, measuring the number of days per week of corticosteroid cream (triamcinolone) use. Secondary outcomes include the difference in use of corticosteroid cream measured in grams, differences in clinical efficacy, quality of life (QoL), microbial composition (includi23ng S. aureus) between the Staphefekt and the placebo group, and the safety and tolerability. DISCUSSION: The results of this trial will provide data about the effect of long-term anti-staphylococcal therapy with Staphefekt on corticosteroid use, clinical symptoms and QoL in patients with moderate to severe AD. Additional data about growth characteristics of the skin microbiome, including S. aureus, will give insight into the role of the microbiome as a factor in the pathophysiology of AD. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02840955 . Registered on 11 July 2016.

The Safety and Tolerability of Lumbrokinase DLBS1033 in Healthy Adult Subjects.

BACKGROUND: This study was aimed to evaluate the safety and tolerability of Lumbrokinase DLBS1033 in healthy adult subjects. METHODS: This was a 2-arm, randomized, double-blind, placebo-controlled, cross-over study over 14 days of treatment with DLBS1033 490 mg 3 times daily. Eligible subjects were enrolled at Period 1 and allocated to receive either test drug or placebo, and underwent a clinical assessment including vital signs, electrocardiography, laboratory examination (hemostasis parameters, routine hematology, liver and renal function), the presence of hemorrhagic symptoms and allergic reactions. Afterwards, they went on to a 2-week washout period, and then were crossed-over to receive the alternate drug at Period 2. The procedure of Period 1 was repeated in the same manner with the alternate drug at Period 2. RESULTS: Of 20 subjects enrolled, one subject was lost to follow-up on Evaluation Day-14 of Period 2. Bleeding risk was relatively low as demonstrated by insignificant differences in hemostasis parameters between DLBS1033 and Placebo. Neither were there significant differences between DLBS1033 and Placebo in terms of hematological parameter, each blood chemistry parameter (liver function, renal function, lipid profile, fasting blood glucose), abnormality proportions of urine test, stool occult blood, and ECG interpretation. There were no hemorrhagic symptoms (petechiae, epistaxis, hematoma) and allergic reactions encountered by study subjects during the treatment with DLBS1033 and Placebo. MAJOR CONCLUSION: DLBS1033 given at the dose of 490 mg 3 times daily was safe and tolerable in healthy adults.

Safety and efficacy of dispase and plasmin in pharmacologic vitreolysis.

PURPOSE: To evaluate the safety and efficacy of dispase and plasmin when inducing posterior vitreous detachment (PVD) by intravitreous injection in rabbit eyes. METHODS: Forty-eight young pigmented rabbits were randomized into six groups. Groups 1 and 5 received 0.025 U dispase in test eyes; group 2, 0.1 U dispase; groups 3 and 6, 1 U plasmin; and group 4, 4 U plasmin. All groups received PBS in control eyes. Groups 5 and 6 were euthanatized 15 minutes after surgery for ocular histologic examination. The remaining groups (groups 1-4) received indirect ophthalmoscope and biomicroscopy 15 and 30 minutes; 1, 2, and 8 hours; and 1, 3, and 7 days after surgery. Ultrasonography and electroretinogram were performed 1 hour and 1 and 7 days after surgery. The eyes then were examined by scanning and transmission electron microscopy. RESULTS: Partial or complete PVDs were observed in the eyes that received dispase and plasmin, confirmed by the results of scanning electron microscopy. Light microscopy showed inflammation in both dispase- and plasmin-treated eyes of groups 5 and 6. However, whereas in plasmin-treated eyes the ERG and cell ultrastructure showed no significant changes, in dispase-treated eyes, the amplitudes of ERG showed a significant reduction from baseline and ultrastructural damage to the retina was detected by transmission electron microscopy. Cell damage, preretinal hemorrhage, and cataract were also observed in these eyes. No changes were observed in the control eyes. CONCLUSIONS: Intravitreal injection of dispase at 0.025 U or more can induce PVD, but it is not safe. Plasmin (1-4 U) is safer, except for the potential risk of inducing intraocular inflammation.

Environmental contributions to the allergic asthma epidemic.

Current data overwhelmingly document the existence of a worldwide asthma epidemic, although individual studies remain controversial. The epidemic is thought to involve primarily persons with allergic asthma, and many diverse theories, based on an immunopathologic understanding of disease, have recently emerged to explain this involvement. In the context of recent insights into the immune basis of experimental asthma, we discuss in this review the leading asthma epidemic theories, including a new theory based on inhaled environmental proteases. Although no single theory may yet be fully embraced, there exists substantial hope that a unifying mechanism for the epidemic will be revealed through additional research.

Dissociation of retinal ganglion cells without enzymes.

We describe here methods for dissociating retinal ganglion cells from adult goldfish and rat without proteolytic enzymes, and show responses of ganglion cells isolated this way to step-wise voltage changes and fluctuating current injections. Taking advantage of the laminar organization of vertebrate retinas, photoreceptors and other cells were lifted away from the distal side of freshly isolated goldfish retinas, after contact with pieces of membrane filter. Likewise, cells were sliced away from the distal side of freshly isolated rat retinas, after these adhered to a membrane filter. The remaining portions of retina were incubated in an enzyme-free, low Ca2+ solution, and triturated. After aliquots of the resulting cell suspension were plated, ganglion cells could be identified by dye retrogradely transported via the optic nerve. These cells showed no obvious morphological degeneration for several days of culture. Perforated-patch whole-cell recordings showed that the goldfish ganglion cells spike tonically in response to depolarizing constant current injections, that these spikes are temporally precise in response to fluctuating current injections, and that the largest voltage-gated Na+ currents of these cells were larger than those of ganglion cells isolated with a neutral protease.

Influence of a complementary treatment with oral enzymes on patients with colorectal cancers--an epidemiological retrolective cohort study.

PURPOSE: To evaluate the impact of postoperative treatment with an oral enzyme (OE) preparation given complementary to an antineoplastic therapy in patients with all stages of colorectal cancer. METHODS: The design of this epidemiological study was a retrolective cohort analysis with parallel groups. Design and conduct of the study were performed to current standards for prospective, controlled clinical trials. Of a cohort of 1,242 patients with colorectal cancer (documented in 213 centres), 616 had received complementary treatment with OE (182 OE only, 405 other complementary drugs, 29 protocol violators) and 626 had not received OE (368 control only, 229 other complementary drugs, 29 protocol violators). Of 1,162 patients who had undergone primary surgery, 526 received adjuvant chemotherapy and 218 radiotherapy. The median follow-up time for the OE group was 9.2 months and for the control group 6.1 months. The primary test criterion of efficacy for OE treatment was the multivariate effect size of the changes from baseline of the disease- and therapy-associated signs and symptoms (nausea, vomiting, changes in appetite, stomach pain or stomach disorder, tiredness, depression, memory or concentration disorder, sleep disturbance, dizziness, irritability, dyspnoea at rest, dyspnoea during activity, headache, tumour pain, cachexia, skin disorders and infections). Tumour-related events, e.g. death, were evaluated by the number of events observed and time to event. Safety of treatment with OE was analysed in terms of number and severity of adverse events, their duration, treatment and outcome. RESULTS: A significant reduction in disease-associated signs and symptoms was observed in patients treated with OE alone, but not in those receiving OE in addition to other complementary treatments. Adverse reactions to chemo- and radiotherapy were diminished in all patients receiving OE. Analysis of survival did not demonstrate a reduced number of deaths in the OE group. However, a trend to prolongation of survival was demonstrated, particularly in the patients with disease stage Dukes' D, in the subgroup receiving OE in addition to other complementary treatments. Similar but less-pronounced trends were observed for disease stages Dukes' B and C. In the OE group, 21 of 616 patients (3.4%) experienced OE-associated adverse reactions, all of them mild to moderate gastrointestinal symptoms. CONCLUSION: Complementary treatment of colorectal cancer patients with OE improves their quality of life by reducing both the signs and symptoms of the disease and the adverse reactions associated with adjuvant antineoplastic therapies. This epidemiological retrolective cohort analysis provides evidence that patients may also benefit by a prolongation of survival time. OE were generally well tolerated.

Impact of complementary oral enzyme application on the postoperative treatment results of breast cancer patients--results of an epidemiological multicentre retrolective cohort study.

PURPOSE: [corrected] To evaluate the impact of postoperative treatment with an oral enzyme (OE) preparation given complementary to an antineoplastic therapy in patients with breast cancer. METHODS: The design of this epidemiological study was a retrolective cohort analysis with parallel groups. Design and conduct of the study were performed to current standards for prospective, controlled clinical trials. A cohort of 2,339 breast cancer patients undergoing surgical intervention and radio-, chemo- or hormonal therapy were studied in 216 centres. Of the 2,339 patients, 1,283 received complementary treatment with OE and 1,056 did not receive OE. Patients with other complementary medications were excluded and the final analysis was performed with the data from 649 patients, of whom 239 (37%) were additionally treated with OE (test group) and 410 (63%) without OE (control group). The median follow-up time for the test group was 485 days and for the control group 213 days. The primary endpoint of the study was to determine whether complementary treatment with OE can reduce typical disease- or therapy-associated signs and symptoms (gastrointestinal symptoms, mental symptoms, dyspnoea, headache, tumour pain, cachexia, skin disorders, infections, and side effects associated with the antineoplastic therapy) in patients with breast cancer. Imbalances for causal effects (covariates) were adjusted for by means of the propensity score. Outcome analysis was performed by estimating the linear regression between change in symptom score and propensity score with all data and using this regression line to calculate the change in symptom score which would be expected for each patient. Tumour-associated events (recurrence, metastasis, and death) were evaluated in terms of the number of events observed and time to event. The safety of treatment with OE was analysed in terms of the number and severity of adverse events, their duration, treatment and outcome. RESULTS: For all symptoms except tumour pain, the adjusted mean improvement in symptom scores was larger in the test group than in the control group. The adjusted difference was statistically significant for all symptoms, except tumour pain and infections. The results show that the typical disease- and therapy-associated signs and symptoms in patients on complementary therapy with OE during postoperative treatment were significantly less. For 75% of the test group and 55% of the control group the physician recorded "no signs and symptoms". A clear reduction in the side effects of radiotherapy and chemotherapy was documented in 74% of the test group and 55% of the control group. Analysis of survival, recurrence, and metastasis demonstrated a reduced number of events in the test group. There was evidence of a beneficial influence of OE on time to event, although the median observation time was too short in these breast cancer patients to draw definite conclusions. The safety component was judged in 98% of the test group and 76% of the control group as "very good" or "good". In the total sample of 2,339 patients, the rate of OE-associated adverse reactions was 3.2%. All side effects were mild to moderate gastrointestinal symptoms. CONCLUSION: Complementary treatment of breast cancer patients with OE improves the quality of life by reducing signs and symptoms of the disease and the side effects of adjuvant antineoplastic therapies. This epidemiological retrolective cohort analysis provides evidence that the patients may also gain benefit by a prolongation of the time to event for cancer recurrence, metastasis and survival. OE was generally well tolerated.

Retrolective cohort study of an additive therapy with an oral enzyme preparation in patients with multiple myeloma.

PURPOSE: To evaluate the impact of an additive therapy with an oral enzyme (OE) preparation given for more than 6 months additionally to standard combination chemotherapy (vincristine/melphalan/cyclophosphamide/prednisone (VMCP)- or methylprednisolone/ vincristine/CCNU/cyclophosphamide/melphalan (MOCCA)-regimen) in the primary treatment of patients with multiple myeloma stages I-III. METHODS: A cohort of 265 patients with multiple myeloma stages I-III was consecutively treated at our institution in two parallel groups (control group (n = 99): chemotherapy +/-OE for less than 6 months; OE-group (n = 166): chemotherapy + OE for more than 6 months). The median follow-up time in the stages I, II, and III for the OE-group was 61, 37, and 46.5 months, respectively; for the control group the respective values were 33, 51.5, and 31.5 months. The primary endpoint of the study was disease-specific survival. Secondary endpoints were response to therapy, duration of first response and side effects. The chosen method for evaluation was the technique of a retrolective cohort analysis with a concurrent control group. Survival analysis was performed by the Kaplan-Meier method and multivariate analysis was done with the Cox proportional hazards model. RESULTS: Significantly higher overall response rates and longer duration of remissions were observed in the OE-group. Primary responders showed a longer mean survival time than non-responders. Additive therapy with OE given for more than 6 months decreased the hazard of death for patients at all stages of disease by approximately 60%. Observation time was not long enough to estimate the median survival for patients at stages I and II; for stage III patients it was 47 months in the control group versus 83 months for the patients treated with OE (P = 0.0014) which means a 3-year gain of survival time. Significant prognostic factors for survival, in the Cox regression analysis, were stage of disease and therapy with OE. The OE-therapy was generally well tolerated (3.6% of patients with mild to moderate gastrointestinal symptoms). CONCLUSION: OEs represent a promising new additive therapy in multiple myeloma which will be further evaluated in a randomized phase III trial in the USA.

Complications of chemonucleolysis for lumbar disc disease.

In 13,700 patients who received one or more lumbar disc injections of chymopapain, 401 complications, adverse reactions, and delayed untoward events were recorded, including eight deaths. The deaths were secondary to anaphylaxis, pulmonary embolism, discitis with subacute bacterial endocarditis, ruptured abdominal aortic aneurysms (two patients), encephalitis (of unknown etiology), and myocardial infarction. Of these, the deaths secondary to anaphylaxis and discitis with subacute bacterial endocarditis can be attributed directly to the procedure of chemonucleolysis.

Hemorrhagic complications after the lumbar injection of chymopapain.

There are few reports of hemorrhagic central nervous system complications after chymopapain injection in humans. Two patients are reported who developed hemorrhagic complications after the lumbar injection of chymopapain. The first developed a hemorrhagic encephalomyelopathy followed by clinically suspected acute arachnoiditis, which responded to high doses of dexamethasone. The second patient developed subarachnoid hemorrhage secondary to vertebral artery aneurysm rupture after the injection of chymopapain.

Multiple cerebral hemorrhages following chymopapain chemonucleolysis. Case report.

A case of multiple cerebral hemorrhages following chymopapain chemonucleolysis is reported. The authors believe the probable etiology was intrathecal extravasation of chymopapain after injection of the drug into a lumbar disc space.

Peripheral nerve injury by chymopapain injection.

Chymopapain injected into the intervertebral disc space (chemonucleolysis) has been used clinically in patients with disc disease with success. Neurosurgical complications secondary to the procedure have, however, been reported. In this study, the authors have investigated the possible neurotoxic effect of chymopapain on the peripheral nerve in rat and primate models. While the extrafascicular injection caused no nerve fiber damage, the intrafascicular injection caused dose-related nerve fiber damage in both species.

Inadequacies and hazards of chymopapain injections as treatment for intervertebral disc disease.

Chymopapain chemonucleolysis was performed on 100 patients with primary lumbar intervertebral disc disease. The results were compared with those of 174 patients who underwent laminotomy, foraminotomy, and discectomy. Primary lumbar intervertebral disc disease was arbitrarily divided into degenerative, complex, previous surgical, and simple disc syndromes. No difference was seen between chemonucleolysis and surgery in the first three divisions; between 55 percent and 60 per cent of patients responded successfully to treatment. In the simple disc division 89 per cent of the surgical and 60 per cent of the chemonucleolysis patients had successful results.

Anaphylactic reactions following the intradiscal injection of chymopapain under local anesthesia.

During the twelve-year period from January 1, 1970, through December 31, 1981, 4,282 patients with the diagnosis of herniated nucleus pulposus were treated by intradiscal injection of chymopapain under local anesthesia. Fifteen (0.35 per cent) of these patients sustained an anaphylactic reaction as defined by us. Twelve patients had subjective early warning signs before their blood pressure decreased, including a total-body burning or tingling sensation (five patients), a general feeling of ill health (four patients), and diffuse pruritus (three patients). Profound hypotension without subjective warning symptoms was the first indication of anaphylaxis in three patients. In all patients, hypotension requiring vigorous treatment was the life-threatening clinical manifestation of anaphylaxis, but respiratory distress severe enough to require endotracheal intubation did not occur. There were no deaths or known sequelae. Ten of the fifteen patients were women. Review of the medical histories of the fifteen patients and follow-up telephone interviews did not identify any other pre-disposing factor for the anaphylaxis. Twelve of the fifteen patients obtained complete relief of the symptoms of disc herniation. The advantage of the use of local rather than general anesthesia for chymopapain injection is that the patient remains responsive and can give an early warning of the subjective symptoms of anaphylaxis if they appear. This potential for early diagnosis allows early and aggressive treatment with intravenous fluids, epinephrine, steroids, and antihistamines, which can be effective in preventing death or permanent sequelae. In our experience, general anesthesia and routine endotracheal intubation are not necessary for intradiscal injection of chymopapain.

Chymopapain-induced hypersensitivity following chemonucleolysis.

This study describes the changes in chymopapain-specific IgE antibody levels in patients following chemonucleolysis with Chymodiactin. Using the ChymoFAST method, chymopapain-specific IgE values were studied in 91 patients prior to and for 2 months post-Chymodiactin chemonucleolysis. A total of 8.8% (17/91) developed IgE levels greater than or equal to 0.06 IU/ml. Those patients with detectable IgE levels prior to chemonucleolysis were more likely than those with nondetectable preinjection levels (36.4% versus 4%) to develop chymopapain-specific IgE levels greater than or equal to 0.06 IU/ml.

Chymodiactin postmarketing surveillance. Demographic and adverse experience data in 29,075 patients.

Postmarketing surveillance data on 29,075 patients who received Chymodiactin (Smith Laboratories' formulation of chymopapain) intradiscal injections for a herniated lumbar intervertebral disc are summarized and tabulated. The serious adverse reactions reported include death, anaphylaxis, paraplegia, and discitis. Similar problems also have been reported for Discase (Baxter-Travenol's formulation of chymopapain). Of 11 deaths reported following Chymodiactin administration, only 3 appear to be related to the drug or procedure. Two of these three were due to anaphylaxis and the third to bacterial discitis with resultant meningitis. Paraplegia appeared to be primarily due to needle trauma or injection of contrast agent and enzyme into the subarachnoid space. Careful patient selection and needle placement are essential for avoiding serious problems.

Intravitreal injection of dispase causes retinal hemorrhages in rabbit and human eyes.

PURPOSE: To check the effects of intravitreally injected dispase in the vitreo-retinal region. METHODS: Dispase, 0.05 to 2.5 units dissolved in 100 microl of phosphate-buffered saline, was injected into the midvitreous of rabbits which were killed from 15 to 120 min afterwards. The enzyme was also injected into four human eyes of patients with orbital tumors 15 min before enucleation during orbital exenteration surgery. The eyes were examined in vivo as well as by light and electron microscopy. RESULTS: Hemorrhages were detected by fundus observations and confirmed by microscopical analysis in nearly all rabbits and in half of the human eyes. The red blood cells were observed in the vitreous and retina. Breaches in the inner limiting membrane were visualized in human eyes and ruptures of small blood vessels in rabbit eyes. In spite of that, vitreous detachment was not verified. In fact, the cortical-vitreous collagen-fibril network was conspicuous on scanning electron micrographs. CONCLUSIONS: Retinal hemorrhages were evident as early as 11 min after injection. It is suggested that this enzyme degraded selectively basement membrane components without affecting other proteins involved in the vitreous-retinal junction.

Two anaphylactic deaths after chemonucleolysis.

Chemonucleolysis is a procedure for treatment of low back pain due to discogenic disease in which the drug chymopapain is injected into lumbar disks to produce chemical dissolution of the nucleus pulposus. More than 15,000 cases have been treated by chemonucleolysis world-wide. Anaphylaxis after the injection of chymopapain occurs in about 1% of such cases. The two cases described in this paper are the only known deaths due to anaphylaxis. Both patients suddenly became hypotensive after injection of chymopapain into a disk. One patient died shortly after this, whereas the second patient died of the complications of prolonged shock.