Ethical argument for establishing good manufacturing practice for phage therapy in the UK.

Antimicrobial resistance (AMR) poses an increasing threat to patient care and population health and there is a growing need for novel therapies to tackle AMR. Bacteriophage (phage) therapy is a re-emerging antimicrobial strategy with the potential to transform how bacterial infections are treated in patients and populations. Currently, in the UK, phages can be used as unlicensed medicinal products on a 'named-patient' basis. We make an ethical case for why it is crucially important for the UK to invest in Good Manufacturing Practice (GMP) for both ongoing unlicensed and future licensed phage therapy. Access to phages produced to GMP (GMP phages) will ensure effective patient care and better outcomes as well as health systems benefits. The UK also has the potential to become a global leader in the timely and cost-efficient manufacturing and supply of a therapy that meets internationally recognised standards.

The Safety and Efficacy of Phage Therapy for Infections in Cardiac and Peripheral Vascular Surgery: A Systematic Review.

New approaches to managing infections in cardiac and peripheral vascular surgery are required to reduce costs to patients and healthcare providers. Bacteriophage (phage) therapy is a promising antimicrobial approach that has been recommended for consideration in antibiotic refractory cases. We systematically reviewed the clinical evidence for phage therapy in vascular surgery to support the unlicensed use of phage therapy and inform future research. Three electronic databases were searched for articles that reported primary data about human phage therapy for infections in cardiac or peripheral vascular surgery. Fourteen reports were eligible for inclusion, representing 40 patients, among which an estimated 70.3% of patients (n = 26/37) achieved clinical resolution. A further 10.8% (n = 4/37) of patients showed improvement and 18.9% (n = 7/37) showed no improvement. Six of the twelve reports that commented on the safety of phage therapy did not report adverse effects. No adverse effects documented in the remaining six reports were directly linked to phages but reflected the presence of manufacturing contaminants or release of bacterial debris following bacterial lysis. The reports identified by this review suggest that appropriately purified phages represent a safe and efficacious treatment option for infections in cardiac and peripheral vascular surgery.

Phage therapy: Awareness and demand among clinicians in the United Kingdom.

Bacterial resistance or tolerance to antibiotics is costly to patients and healthcare providers. With the impact of antibiotic resistance forecast to grow, alternative antimicrobial approaches are needed to help treat patients with antibiotic refractory infections and reduce reliance upon existing antibiotics. There is renewed interest in bacteriophage (phage) therapy as a promising antimicrobial strategy. We therefore performed the first multi-specialty survey about phage therapy and the first such survey among clinicians in the United Kingdom. An anonymous 10-question survey of clinicians from medical and surgical specialties in two Scottish Health Boards was performed. The 90 respondents spanned 26 specialties and were predominantly consultants (73.3%). The respondents were concerned about antibiotic resistance in their clinical practice; 83 respondents estimated having seen 711 patients in the last 12 months whose infections were refractory to antibiotics (delaying or preventing resolution). Over half (58.8%) of the respondents had previously heard of phage therapy. Staphylococci, Pseudomonas and E. coli were identified as the highest cross-specialty priorities for the development of phage therapy. Together, 77 respondents estimated seeing 300 patients in the last 12 months for whom phage therapy may have been appropriate (an average of 3.9 patients per clinician). Most respondents (71.1%, n = 90) were already willing to consider using phage therapy in appropriate cases. Additional comments from the respondents affirmed the potential utility of phage therapy and highlighted a need for more information. The results of this survey demonstrate substantial demand for and willingness to use phage therapy in appropriate cases, both from individual clinicians and across specialties. Demand from a wide range of specialties illustrates the broad clinical utility of phage therapy and potential scope of impact. Widening access to phage therapy could deliver substantial clinical and financial benefits for patients and health authorities alike.

Conserved 5-methyluridine tRNA modification modulates ribosome translocation.

While the centrality of post-transcriptional modifications to RNA biology has long been acknowledged, the function of the vast majority of modified sites remains to be discovered. Illustrative of this, there is not yet a discrete biological role assigned for one the most highly conserved modifications, 5-methyluridine at position 54 in tRNAs (m 5 U54). Here, we uncover contributions of m 5 U54 to both tRNA maturation and protein synthesis. Our mass spectrometry analyses demonstrate that cells lacking the enzyme that installs m 5 U in the T-loop (TrmA in E. coli , Trm2 in S. cerevisiae ) exhibit altered tRNA modifications patterns. Furthermore, m 5 U54 deficient tRNAs are desensitized to small molecules that prevent translocation in vitro. This finding is consistent with our observations that, relative to wild-type cells, trm2 Δ cell growth and transcriptome-wide gene expression are less perturbed by translocation inhibitors. Together our data suggest a model in which m 5 U54 acts as an important modulator of tRNA maturation and translocation of the ribosome during protein synthesis.

Phage Therapy for Diabetic Foot Infection: A Case Series.

PURPOSE: Infected diabetic foot ulcers can be difficult to treat and, despite appropriate antibiotic therapy, some diabetic foot infections (DFIs) require amputation. Bacteriophages (phages) are viruses that infect and kill bacteria. Phage therapy has been repeatedly used to successfully treat DFIs and other chronic wounds. METHODS: This article reports the provision of topical adjunctive anti-staphylococcal phage therapy to 10 patients with DFI at high risk of amputation at two UK hospitals as part of clinical care; tolerability and efficacy were clinically assessed. FINDINGS: The opinion of the experienced clinical teams caring for these patients was that 9 of the 10 patients appeared to benefit from adjunctive phage therapy. No adverse effects were reported by clinicians or patients. In 6 of 10 patients the clinical impression was that phage therapy facilitated clinical resolution of infection and limb salvage. Resolution of soft tissue infection was observed in a 7th patient but unresolved osteomyelitis required amputation. An 8th patient demonstrated eradication of Staphylococcus aureus from a polymicrobial infection and a 9th showed signs of clinical improvement before early cessation of phage therapy due to an unrelated event. One patient, with a weakly susceptible S aureus isolate, had no significant response. IMPLICATIONS: This report describes the largest application of phage therapy in the United Kingdom to date and the first application of phage therapy for DFI in the United Kingdom and offers subjective hints toward impressive tolerability and efficacy. Phage therapy has the potential to transform the prevention and treatment of DFIs.

Direct sequencing of total Saccharomyces cerevisiae tRNAs by LC-MS/MS.

Among RNAs, transfer RNAs (tRNAs) contain the widest variety of abundant posttranscriptional chemical modifications. These modifications are crucial for tRNAs to participate in protein synthesis, promoting proper tRNA structure and aminoacylation, facilitating anticodon:codon recognition, and ensuring the reading frame maintenance of the ribosome. While tRNA modifications were long thought to be stoichiometric, it is becoming increasingly apparent that these modifications can change dynamically in response to the cellular environment. The ability to broadly characterize the fluctuating tRNA modification landscape will be essential for establishing the molecular level contributions of individual sites of tRNA modification. The locations of modifications within individual tRNA sequences can be mapped using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). In this approach, a single tRNA species is purified, treated with ribonucleases, and the resulting single-stranded RNA products are subject to LC-MS/MS analysis. The application of LC-MS/MS to study tRNAs is limited by the necessity of analyzing one tRNA at a time, because the digestion of total tRNA mixtures by commercially available ribonucleases produces many short digestion products unable to be uniquely mapped back to a single site within a tRNA. We overcame these limitations by taking advantage of the highly structured nature of tRNAs to prevent the full digestion by single-stranded RNA-specific ribonucleases. Folding total tRNA prior to digestion allowed us to sequence Saccharomyces cerevisiae tRNAs with up to 97% sequence coverage for individual tRNA species by LC-MS/MS. This method presents a robust avenue for directly detecting the distribution of modifications in total tRNAs.

Methylated guanosine and uridine modifications in S. cerevisiae mRNAs modulate translation elongation.

Chemical modifications to protein encoding messenger RNAs (mRNAs) influence their localization, translation, and stability within cells. Over 15 different types of mRNA modifications have been observed by sequencing and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) approaches. While LC-MS/MS is arguably the most essential tool available for studying analogous protein post-translational modifications, the high-throughput discovery and quantitative characterization of mRNA modifications by LC-MS/MS has been hampered by the difficulty of obtaining sufficient quantities of pure mRNA and limited sensitivities for modified nucleosides. We have overcome these challenges by improving the mRNA purification and LC-MS/MS pipelines. The methodologies we developed result in no detectable non-coding RNA modifications signals in our purified mRNA samples, quantify 50 ribonucleosides in a single analysis, and provide the lowest limit of detection reported for ribonucleoside modification LC-MS/MS analyses. These advancements enabled the detection and quantification of 13 S. cerevisiae mRNA ribonucleoside modifications and reveal the presence of four new S. cerevisiae mRNA modifications at low to moderate levels (1-methyguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, and 5-methyluridine). We identified four enzymes that incorporate these modifications into S. cerevisiae mRNAs (Trm10, Trm11, Trm1, and Trm2, respectively), though our results suggest that guanosine and uridine nucleobases are also non-enzymatically methylated at low levels. Regardless of whether they are incorporated in a programmed manner or as the result of RNA damage, we reasoned that the ribosome will encounter the modifications that we detect in cells. To evaluate this possibility, we used a reconstituted translation system to investigate the consequences of modifications on translation elongation. Our findings demonstrate that the introduction of 1-methyguanosine, N2-methylguanosine and 5-methyluridine into mRNA codons impedes amino acid addition in a position dependent manner. This work expands the repertoire of nucleoside modifications that the ribosome must decode in S. cerevisiae. Additionally, it highlights the challenge of predicting the effect of discrete modified mRNA sites on translation de novo because individual modifications influence translation differently depending on mRNA sequence context.

Managing Patient and Clinician Expectations of Phage Therapy in the United Kingdom.

Bacteriophage (phage) therapy is a promising alternative antimicrobial approach which has the potential to transform the way we treat bacterial infections. Phage therapy is currently being used on a compassionate basis in multiple countries. Therefore, if a patient has an antibiotic refractory infection, they may expect their clinician to consider and access phage therapy with the hope of improvement. The expectations of clinicians may be similar and may also include expectations around data collection. However, there are multiple biological and practical barriers to fulfilling patient and clinician expectations. While it is possible to access phage therapy, the path to acquisition is not straightforward and expectations therefore need to be managed appropriately to avoid raising false hope and undermining confidence in phage therapy. Phage scientists have an important contribution to make in educating clinicians and the broader public about phage therapy. However, it is clinicians that are responsible for managing the expectations of their patients and this relies on clear communication about the barriers and limitations.

The Future of Clinical Phage Therapy in the United Kingdom.

Bacteriophage (phage) therapy is a promising alternative antimicrobial strategy with the potential to transform the way bacterial infections are treated. In the United Kingdom, phages are classed as a biological medicine. Although no phages are licensed for UK use, they may be used as unlicensed medicinal products where licensed alternatives cannot meet a patient's clinical needs. In the last 2 years, 12 patients in the UK have received phage therapy, and there is burgeoning clinical interest. Currently, clinical phage provision in the UK is ad hoc and relies upon networking with international sources of phages. The provision of phage therapy in the UK will not progress beyond an increasing number of ad hoc cases until an onshore sustainable and scalable source of well-characterised phages manufactured in accordance with Good Manufacturing Practice (GMP) is established. Here, we present an exciting new collaboration between UK Phage Therapy, the Centre for Phage Research at University of Leicester, CPI, and Fixed Phage. These partners, and others as we develop, will establish sustainable, scalable, and equitable phage therapy provision in the UK. We set out a vision for how phage therapy will be integrated into the NHS and healthcare more broadly, including the complementarity between licensed (cocktail) and unlicensed (personalised) phage preparations. Key elements of phage therapy infrastructure in the UK will be GMP phage manufacturing, a national phage library, and a national clinical phage centre. Together, this infrastructure will support NHS microbiology departments to develop and oversee phage therapy provision across the UK. As it will take time to deliver this, we also describe considerations for clinicians seeking to use unlicensed phage therapy in the interim. In summary, this review sets out a roadmap for the delivery of clinical phage therapy to the UK, the benefits of which we hope will reverberate for patients for decades to come.

Nuclease P1 Digestion for Bottom-Up RNA Sequencing of Modified siRNA Therapeutics.

siRNA therapeutics provide a selective and powerful approach to reduce the expression of disease-causing genes. For regulatory approval, these modalities require sequence confirmation which is typically achieved by intact tandem mass spectrometry sequencing. However, this process produces highly complex spectra which are difficult to interpret and typically results in less than full sequence coverage. We sought to develop a bottom-up siRNA sequencing platform to ease sequencing data analysis and provide full sequence coverage. Analogous to bottom-up proteomics, this process requires chemical or enzymatic digestion to reduce the oligonucleotide length down to analyzable lengths, but siRNAs commonly contain modifications that inhibit the degradation process. We tested six digestion schemes for their feasibility to digest the 2' modified siRNAs and identified that nuclease P1 provides an effective digestion workflow. Using a partial digestion, nuclease P1 provides high 5' and 3' end sequence coverage with multiple overlapping digestion products. Additionally, this enzyme provides high-quality and highly reproducible RNA sequencing no matter the RNA phosphorothioate content, 2'-fluorination status, sequence, or length. Overall, we developed a robust enzymatic digestion scheme for bottom-up siRNA sequencing using nuclease P1, which can be implemented into existing sequence confirmation workflows.

Sequence-specific capture and concentration of viral RNA by type III CRISPR system enhances diagnostic.

Type-III CRISPR-Cas systems have recently been adopted for sequence-specific detection of SARS-CoV-2. Here, we repurpose the type III-A CRISPR complex from Thermus thermophilus (TtCsm) for programmable capture and concentration of specific RNAs from complex mixtures. The target bound TtCsm complex generates two cyclic oligoadenylates (i.e., cA3 and cA4) that allosterically activate ancillary nucleases. We show that both Can1 and Can2 nucleases cleave single-stranded RNA, single-stranded DNA, and double-stranded DNA in the presence of cA4. We integrate the Can2 nuclease with type III-A RNA capture and concentration for direct detection of SARS-CoV-2 RNA in nasopharyngeal swabs with 15 fM sensitivity. Collectively, this work demonstrates how type-III CRISPR-based RNA capture and concentration simultaneously increases sensitivity, limits time to result, lowers cost of the assay, eliminates solvents used for RNA extraction, and reduces sample handling.

The practical, ethical and legal reasons why patients should not be transferred between NHS trusts for phage therapy.

Bacteriophages (phages) are naturally occurring viruses of bacteria that have a long history of use as antimicrobials, known as phage therapy. The antibiotic resistance crisis has driven renewed interest in phage therapy, which has been used on an unlicensed compassionate basis in various Western contexts. The option to use unlicensed medicines exists to allow clinicians to respond to genuine clinical needs arising in their own patients. However, in the UK some clinicians may, in the absence of suitable patients of their own, seek to transfer patients from other NHS trusts into their own Trust. This article sets out why patient transfer is not necessary and the practical, ethical and legal reasons why patients should not be transferred between NHS Trusts for phage therapy. Phage preparations should always be transported to the patient and the patient treated in the Trust in which they would have received care in the absence of phage. We enclose suggested best practice guidelines for adoption across the UK that will protect patient safety and safeguard clinicians and Trusts from potential litigation.

The Safety and Efficacy of Phage Therapy: A Systematic Review of Clinical and Safety Trials.

Trials of phage therapy have not consistently reported efficacy. This contrasts with promising efficacy rates from a sizeable and compelling body of observational literature. This systematic review explores the reasons why many phage trials have not demonstrated efficacy. Four electronic databases were systematically searched for safety and/or efficacy trials of phage therapy. Sixteen trials of phage therapy were included, in which 378 patients received phage. These were divided into historical (pre-2000; N = 3; n = 76) and modern (post-2000; N = 13; n = 302) trials. All 13 modern trials concluded that phage therapy was safe. Six of the 13 modern trials were exclusively safety trials. Seven modern trials investigated both safety and efficacy; efficacy was observed in two. Two of three historical trials did not comment on safety, while adverse effects in the third likely reflected the use of phage preparations contaminated with bacterial debris. None of the historical trials contained evidence of efficacy. The evidence from trials is that phage therapy is safe. For efficacy to be observed a therapeutic amount of the right phage(s) must be delivered to the right place to treat infections containing enough susceptible bacterial cells. Trials that have not demonstrated efficacy have not fulfilled one or more elements of this principle.

Sequence-specific capture and concentration of viral RNA by type III CRISPR system enhances diagnostic.

Type-III CRISPR-Cas systems have recently been adopted for sequence-specific detection of SARS-CoV-2. Here, we make two major advances that simultaneously limit sample handling and significantly enhance the sensitivity of SARS-CoV-2 RNA detection directly from patient samples. First, we repurpose the type III-A CRISPR complex from Thermus thermophilus (TtCsm) for programmable capture and concentration of specific RNAs from complex mixtures. The target bound TtCsm complex primarily generates two cyclic oligoadenylates (i.e., cA3 and cA4) that allosterically activate ancillary nucleases. To improve sensitivity of the diagnostic, we identify and test several ancillary nucleases (i.e., Can1, Can2, and NucC). We show that Can1 and Can2 are activated by both cA3 and cA4, and that different activators trigger changes in the substrate specificity of these nucleases. Finally, we integrate the type III-A CRISPR RNA-guided capture technique with the Can2 nuclease for 90 fM (5x104 copies/ul) detection of SARS-CoV-2 RNA directly from nasopharyngeal swab samples.

The microbiology of diabetic foot infections: a meta-analysis.

BACKGROUND: Diabetic foot ulcers are a common complication of poorly controlled diabetes and often become infected, termed diabetic foot infection. There have been numerous studies of the microbiology of diabetic foot infection but no meta-analysis has provided a global overview of these data. This meta-analysis aimed to investigate the prevalence of bacteria isolated from diabetic foot infections using studies of any design which reported diabetic foot infection culture results. METHODS: The Medline, EMBASE, Web of Science and BIOSIS electronic databases were searched for studies published up to 2019 which contained microbiological culture results from at least 10 diabetic foot infection patients. Two authors independently assessed study eligibility and extracted the data. The main outcome was the prevalence of each bacterial genera or species. RESULTS: A total of 112 studies were included, representing 16,159 patients from which 22,198 microbial isolates were obtained. The organism most commonly identified was Staphylococcus aureus, of which 18.0% (95% CI 13.8-22.6%; I2 = 93.8% [93.0-94.5%]) was MRSA. Other highly prevalent organisms were Pseudomonas spp., E. coli and Enterococcus spp. A correlation was identified between Gross National Income and the prevalence of Gram positive or negative organisms in diabetic foot infections. CONCLUSION: The microbiology of diabetic foot infections is diverse, but S. aureus predominates. The correlation between the prevalence of Gram positive and negative organisms and Gross National Income could reflect differences in healthcare provision and sanitation. This meta-analysis has synthesised multiple datasets to provide a global overview of the microbiology of diabetic foot infections that will help direct the development of novel therapeutics.

Anatomical variations of the circle of Willis and their prevalence, with a focus on the posterior communicating artery: A literature review and meta-analysis.

The circle of Willis is an anastomotic network of arteries surrounding the base of the brain, providing collateral circulation to prevent ischemia. It has, however, long been established that it exhibits considerable anatomical variation when compared to Thomas Willis' originally described circle. This study aimed primarily to determine an accurate prevalence of the variation of the circle of Willis in the general population and the prevalence of common posterior communicating artery variations. Additional aims were to explain why such a wide range of reported variations exist, and whether different types of studies report significantly different prevalence of variation. A comprehensive literature search identified 764 papers. A three-phase screening process was undertaken, involving a critical analysis of papers, and a total of 33 papers were selected for analysis and literature review. A descriptive statistics test with bootstrap was performed to estimate the average prevalence of variations. The estimated prevalence of general variation, unilateral, and bilateral posterior communicating artery hypoplasia or aplasia was 68.22 ± 14.32%, 19.45 ± 8.63%, and 22.83 ± 14.58%, respectively. Over half of the population exhibit a circle of Willis with some form of variation. To provide a more accurate estimation for the prevalence of variations, a universal classification system needs to be established, collating all the work from high-quality studies, to provide a comprehensive database of the circle's variations. Knowing the prevalence of variations and how they can impact neurosurgical approaches or patterns of ischemic pathology can be crucial in providing effective patient care.

Patient perceptions of phage therapy for diabetic foot infection.

Infections of diabetic foot ulcers are common, generally recalcitrant and often complicated by antibiotic resistance. Alternative antimicrobial strategies are needed. Phage therapy is a promising alternative that is being rediscovered. Despite phage therapy's 100-year history, there have been no investigations into patient thoughts and concerns. This study aimed to explore patient awareness of and concern about antibiotic resistance and gain insight into the perceptions of phage therapy among a patient group that could potentially benefit from phage therapy. Patients with an active or resolved (healed or amputated) diabetic foot ulcer were eligible to participate. A survey was distributed digitally to eligible patients across Scotland via the NHS Research Scotland Diabetes Network and hard copies were available in diabetic foot clinics at the Royal Infirmary of Edinburgh and Queen Elizabeth University Hospital, Glasgow. A focus group of five survey respondents was held in Glasgow. Fifty-five survey responses were obtained. There was a high level of awareness (76.4%; N = 55) and concern (83.3%; N = 54) about antibiotic resistance. While largely aware of viruses, most patients had not heard of phage or phage therapy. Patients were no more concerned about phage than antibiotic therapy, with most suggesting more information could alleviate any concerns. Patient acceptability of phage therapy was high, a finding confirmed by the focus group. Patients are concerned about antibiotic resistance and supportive of 'new' antimicrobials. We have demonstrated that patients are supportive, enthusiastic and accepting of phage therapy. Although 'Western' phage therapy remains in its infancy, an understanding of patient ideas, concerns and expectations will be important in eventually shaping and successfully reintroducing phage therapy.

The Safety and Efficacy of Phage Therapy for Bone and Joint Infections: A Systematic Review.

Bacterial resistance to antibiotics has catalysed interest in alternative antimicrobial strategies. Bacteriophages (phages) are viruses of bacteria with a long history of successful therapeutic use. Phage therapy is a promising antibacterial strategy for infections with a biofilm component, including recalcitrant bone and joint infections, which have significant social, financial and human impacts. Here, we report a systematic review of the safety and efficacy of phage therapy for the treatment of bone and joint infections. Three electronic databases were systematically searched for articles that reported primary data about human phage therapy for bone and joint infections. Two authors independently assessed study eligibility and performed data extraction. Seventeen reports were eligible for inclusion in this review, representing the treatment of 277 patients. A cautionary, crude, efficacy estimate revealed that 93.1% (n = 258/277) achieved clinical resolution, 3.3% (n = 9/277) had improvement and 3.6% (n = 10/277) showed no improvement. Seven of the nine reports that directly commented on the safety of phage therapy did not express safety concerns. The adverse effects reported in the remaining two were not severe and were linked to the presence of contaminating endotoxins and pre-existing liver pathology in a patient treated with high-titre intravenous phage therapy. Three other reports, from 1940-1987, offered general comments on the safety of phage therapy and documented adverse effects consistent with endotoxin co-administration concomitant with the use of raw phage lysates. Together, the reports identified by this review suggest that appropriately purified phages represent a safe and highly efficacious treatment option for complex and intractable bone and joint infections.

The Safety and Efficacy of Phage Therapy for Superficial Bacterial Infections: A Systematic Review.

Superficial bacterial infections, such as dermatological, burn wound and chronic wound/ulcer infections, place great human and financial burdens on health systems globally and are often complicated by antibiotic resistance. Bacteriophage (phage) therapy is a promising alternative antimicrobial strategy with a 100-year history of successful application. Here, we report a systematic review of the safety and efficacy of phage therapy for the treatment of superficial bacterial infections. Three electronic databases were systematically searched for articles that reported primary data about human phage therapy for dermatological, burn wound or chronic wound/ulcer infections secondary to commonly causative bacteria. Two authors independently assessed study eligibility and performed data extraction. Of the 27 eligible reports, eight contained data on burn wound infection (n = 156), 12 on chronic wound/ulcer infection (n = 327) and 10 on dermatological infections (n = 1096). Cautionary pooled efficacy estimates from the studies that clearly reported efficacy data showed clinical resolution or improvement in 77.5% (n = 111) of burn wound infections, 86.1% (n = 310) of chronic wound/ulcer infections and 94.14% (n = 734) of dermatological infections. Over half of the reports that commented on safety (n = 8/15), all published in or after 2002, did not express safety concerns. Seven early reports (1929-1987), described adverse effects consistent with the administration of raw phage lysate and co-administered bacterial debris or broth. This review strongly suggests that the use of purified phage to treat superficial bacterial infections can be highly effective and, by various routes of administration, is safe and without adverse effects.