Rate of Discovery of New Drugs Assignment Paper

Over the years, the rate of discovery of new drugs has declined significantly, leading to an urge to explore alternative environmental sources apart from the soil, for novel antibiotics. In this study, water samples were collected from different places around Bath and they were screened for antibacterial activity against six test bacteria. A total of 106 isolates were isolated and tested against methicillin-sensitive Staphylococcus aureus (MSSA) to confirm their antagonistic activity. 34 isolates showed promising inhibition and were further tested against bacteria which included MSSA, methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecium (E. faecium), Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa) and Klebsiella pneumoniae (K. pneumoniae) through perpendicular streak test. The supernatant of the isolates was also extracted by centrifugation and assayed for its antibacterial activity. For E. coli-inhibiting isolates, further tests against strains of E. coli with different antibiotic resistance were performed to identify the types of antibiotics produced. 11 active isolates were effective against both Gram-positive and Gram-negative bacteria in the perpendicular streak test. The supernatant of the isolates exhibited minimal antibacterial activity. Following the results of 16S rRNA sequencing, two active isolates belonged to Pseudomonas species while the other three isolates were classified as Bacillus species. Isolate 107 was identified as Bacillus pumilus and it demonstrated the strongest inhibition against MSSA, MRSA, E. faecium and E. coli with a zone of inhibition of 20.8mm, 24.3mm, 16.2mm and 13.6mm respectively. Moreover, isolates 18 and 107 both had strong activity against all strains of E. coli tested, while isolate 71 was only active against four strains of E. coli tested, suggesting that one of the antibiotics produced may be kanamycin. Our findings indicate that aquatic environment is a potent source for the isolation of bioactive microorganism potential for the production of antibacterial compounds.Rate of Discovery of New Drugs Assignment Paper

 

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Clinical drug development is available for study to MSc or PGDip level, over a period of one year full-time or two- five years part time (variable mode).

We have developed a modular postgraduate programme in clinical drug development designed to give you the necessary academic background and specialist skills needed to carry out clinical drug development in a contract research organisation, pharmaceutical industry or health service environment.

If you are a nurse, medical doctor or other health professional working in contract research organisations, the pharmaceutical industry and healthcare, this programme has been designed for you.

The programme will:

Allow you the option to study on a flexible part-time basis, or by distance learning.
Give you an understanding of the regulatory framework underlying clinical research.
Teach you to understand the principles of laboratory methodologies applied to clinical trials.
Give you understanding of the steps involved in developing and implementing new drugs.
Cover the key areas of expertise needed for a successful clinical research programme.
Develop your skills and understanding in clinical microbiology, and will give you a thorough knowledge of associated subjects such as molecular biology.
Why study your MSc in Clinical drug development at Queen Mary?
Barts and the London School of Medicine and Dentistry is comprised of two world renowned teaching hospitals, St Bartholomew’s and The Royal London, which have made, and continue to make, an outstanding contribution to modern medicine. We are one of the top five in the UK for medicine in the 2008 Research Assessment Exercise.Rate of Discovery of New Drugs Assignment Paper

MSc/PGDip in Clinical drug Development is based at The William Harvey Research Institute, the largest university-based pharmacological research institute in the UK. Our success in this area is illustrated by our publications in high-impact journals, accompanied by renewal and additional funding of one Medical Research Council and five Wellcome Programmes, which we lead or support as co-investigators. The Institute has strong links with the pharmaceutical industry both in the UK and abroad.

Our location in the east of London and elsewhere in the East London Gateway will enhance your experience.
Barts and The London is part of Queen Mary, the only university in central London to offer extensive campus-based facilities.
We have modern state-of-the-art buildings alongside more traditional facilities such as our fantastic library.
The modular nature of the programme is designed to fit in with your full-time employment.
Facilities
You will have access to postgraduate only facilities. The Learning Resource centre has 200 networked PCs and is open to students round the clock, there are dedicated workstations for postgraduate students.

You will also have access to Queen Mary’s comprehensive libraries, including the Postgraduate Reading Room, and The British Library can also be accessed as a research resource. There are medical libraries located at the Royal London and St Bart’s hospitals and at the main College campus at Mile End.

The William Harvey Research Institute offers state-of-the art core facilities, including a Genome Centre, a flow cytometry and cell sorting station, and in vivo imaging facilities.

Distance learning students will have access to the college online library facilities. This gives access to a large number of relevant journals. Students will have access to other academic literature and journals in the same way as on-site students.

The drug discovery process is the cornerstone of the pharmaceutical industry. Its purpose is to ensure that a drug or medicinal product is as safe and effective as possible for its indicated use in humans before being authorized for marketing. Even though this process is not perfect, it is still critical to drug approval, because regulatory authorities, such as the U.S. Food and Drug Administration and the Federal Institute of Drugs and Medical Devices in Germany, require successful human clinical trials before a drug, procedure or medical product can receive marketing authority.

Clinical trials involving drugs are the most common; however, clinical trials can involve new medical products, such as blood glucose monitoring devices, new diagnostic procedures and even the discovery of new uses for traditional medicines. Perhaps the best example of the latter is aspirin. It was originally used as an analgesic, but, as a result of clinical trials, is also approved to treat and prevent cardiovascular disease, strokes and heart attacks.

The majority of current clinical trials involve a drug used to treat a specific illness and are thus known as treatment studies. Other types of clinical studies include genetic studies, which investigate the relationship between genes and illnesses, with the goal of developing individual treatments based on an individual’s genetic makeup; epidemiological studies, which attempt to identify the patterns and causes of illnesses in groups of people; and observational studies, which involve the comparison of subjects against a control group.Rate of Discovery of New Drugs Assignment Paper

There are three other types of studies that are often conducted as part of a larger trial but that can also be conducted on their own: pharmacokinetic, pharmacodynamic and pharmacogenomic studies. Pharmacokinetic studies investigate how the body affects a specific drug after administration. This involves four components that can be summarized by the acronym ADME (absorption, distribution, metabolism and excretion). Pharmacodynamic studies investigate how drugs affect the body. Common drug actions studied include stimulating and/or depressing action through direct receptor agonism and downstream effects. Finally, pharmacogenomic studies investigate the influence of genetic variation in patients. The aim of such studies is to develop optimized drug treatments taking into account patients’ genotypes, which maximize efficacy and minimize adverse effects.

While any type of drug can theoretically be tested during a clinical trial, the following four types are the most common:

Small molecule drugs (e.g., aspirin). These usually have a molecular weight between 500 and 600. Thus, the molecules can easily pass through the walls of the stomach and duodenum and enter the bloodstream. These drugs are usually taken orally, but other routes of administration are also possible.
Proteins (e.g., insulin). These drugs are too large to pass through the stomach and duodenum and so must be administered by injections or other suitable means.
Vaccines (e.g., human papilloma virus vaccine).
Nucleic acids (e.g., DNA and small interfering RNA). These drugs seek to selectively stop the expression of a certain gene whose expression causes a specific disease.

Cost and Attrition

A new chemical entity, i.e., a drug that has just been synthesized in the laboratory, can cost over $800 million and take as long as 15 years to develop, sometimes even longer (Adam and Brantner, 2006, in Speid, 2010). This amount includes the cost of development programs that failed and/or were terminated. Not surprisingly, given the major importance of clinical trials to the marketing approval of a drug, the majority of a drug’s development budget is spent on clinical testing.

Millions of dollars are invested into a clinical trial with no guarantee that the drug will ultimately receive marketing authorization. Many drugs begin the drug development process, but many of them fail – a process known as attrition. Approximately 62% of phase II drugs undergo attrition. In addition, approximately 23% of drugs that enter the registration phase will not receive marketing approval. This leaves a very low overall success rate of about 11% (Kola and Landis, 2004, in Speid, 2010).

Stages of Drug Discovery

As this figure shows, the drug discovery process is very involved, and the familiar clinical trial phase is only one component of this process. The stepwise nature of this approach is significant because the number of subjects increases at each phase, as does the probability for more subjects to be harmed or not experience any benefit from the drug being investigated. A drug’s development can be terminated (e.g., by the pharmaceutical company or the authorities) at any point in this process if it has been shown that the drug is neither safe nor effective for its proposed indication in humans.Rate of Discovery of New Drugs Assignment Paper

In the pre-discovery phase, scientists gather as much basic information about the illness as possible in an attempt to understand its underlying cause(s) and potential treatment(s). Then, a drug target, which is a naturally occurring cellular or molecular structure that the drug is intended to act on, is selected and validated.

The next phase is drug discovery, and it can last up to 6 years. Scientists use high-throughput screening to quickly identify antibodies, genes or active compounds that alter a certain biomolecular pathway. Anywhere from 5,000 to 10,000 compounds may be tested during this phase. These results are then used as starting points in the development of the drug. Due to the extremely expensive and long process before a drug can be marketed, usually no more than 5 molecules from the thousands of compounds tested will be the “candidate drugs” studied in clinical trials.

The preclinical testing phase begins once the candidate drug has been identified. During this phase, researchers try to understand how the drug works and determine its safety profile for possible testing in humans. The U.S. Food and Drug Administration, as well as most international authorities, requires thorough testing before the candidate drug(s) can be studied in humans. Currently, the only viable ways this can be done is by animal, in vitro, and in vivo studies. If these tests demonstrate the safety and efficacy of the drug, then the drug can move into the familiar clinical research phase. The preclinical testing phase brings together many scientific disciplines, including physiology, chemistry, biology, biotechnology and statistics.

The next phase in the drug discovery process is the clinical research phase. Due to the fact that the number of subjects increases at each stage, this phase can last anywhere from 6 to 7 years. The primary purposes of phase I trials are to determine the effects of the drug on the human body, assess the dose and safety of the drug, and obtain a safe and optimal dose that is likely to be effective for the proposed indication. These trials are designed to answer the question, “How well is the drug tolerated in small numbers of people?” as these trials typically include between 20 and 100 healthy volunteers. Phase I trials typically last anywhere from several days to several weeks.

The next step in the drug discovery pipeline is phase II, and these trials are designed to observe the efficacy of a drug. They are also called the actual testing of the proof of concept (PoC), i.e., does the drug actually work against the disease it was designed to treat? The frequency of adverse events is also measured during this phase. Phase II trials answer the question, “What is an appropriate dose for the drug to be effective?” Since different treatment regimens were likely studied during a particular drug’s phase I trial, the phase II trial often involves at least one treatment regimen for the drug. Phase II trials usually include between 100 and 250 subjects.

If a drug has succeeded in the first two phases, phase III is the next stage, which typically includes anywhere from 1,000 to 5,000 participants. These trials are designed to determine whether a drug is both safe and effective. In addition, adverse reactions resulting from long-term use of the drug are monitored during this phase. If a drug has advanced to phase III, its study population should be defined in advance by inclusion and exclusion criteria, which is a set of medical criteria determining who can (and cannot) participate in a clinical trial. Phase III trials usually take years to complete. The marketing application is submitted to the national supervising authority during this phase. In the U.S., this form is called a New Drug Application (NDA) and is submitted to the U.S. Food and Drug Administration. In Europe, it is known as a Marketing Authorization Application (MAA).Rate of Discovery of New Drugs Assignment Paper

Phase IV trials, commonly called post-marketing studies because they usually take place after a particular drug has received marketing authorization, are the last step in the drug discovery process. Phase IV trials can take years to complete and can have patients numbering in the hundreds to thousands. They are designed to provide more data about a drug in real-life situations. In addition, special and at-risk populations, such as pregnant women, may be studied during this phase.

Considering the increasing pressures on the industry to reduce clinical time and cost, it is clear the industry must find ways to improve these dismal success rates and speed the course of development. A 10% improvement in cycle time and success rates can reduce the total capitalized cost to bring a new drug to market by $634 million.2

Although the pharmaceutical and biotechnology industry has traditionally been slow to embrace new processes and technologies, effective methods to improve productivity, build pipelines faster, streamline infrastructure, lower costs and shorten time to market are extremely valuable — an industry imperative in today’s marketplace. To achieve the needed efficiency, the traditional linear path to early drug development will no longer suffice.

To meet today’s demands, including more rigorous regulatory expectations, drug sponsors are relying more heavily on integrated development services from outsourcing contractors, such as BioDuro, that have the specialized expertise, technologies and innovations to improve efficiency. BioDuro, a U.S.-owned research and development contractor with laboratories in San Diego, Beijing and Shanghai, is a full-service preclinical research and clinical development service provider that offers discovery and development solutions from lead generation through to dosage form development and manufacturing. This article explains how companies such as BioDuro, a pioneer of integrated drug discovery and development services, saves considerable time and costs by utilizing innovative methodologies.

Overcoming Inefficiencies in Clinical Development

The FDA has issued guidance documents describing specific ways for drug developers to advance the earliest phases of clinical research to evaluate scientific advances discovered in their laboratories much more efficiently.3 Yet the drug development process remains highly inefficient, fraught with delays and rising costs. Late-stage failure for novel compounds is high. Attrition rates are as high as 40%, due to drug metabolism and pharmacokinetic (DMPK) issues alone.4

Often, various steps in the development process are provided by different service contractors in a sequential manner. This traditional pathway of drug development is no longer adequate.

To speed the development pathway, drug development service providers, such as BioDuro, are integrating discovery and development services by conducting interdisciplinary studies in areas such as formulation development and DMPK. BioDuro, a hybrid contract research and development & manufacturing organization (CRO-CDMO), takes a multidisciplinary approach, integrating project management throughout full development services in a seamless transition between processes, utilizing the same team of experts in all contributing areas. Its combined team of experts provide solutions in discovery chemistry, biology, DMPK, pharmacology, formulation development and cGMP drug product manufacturing.

Incorporating Early DMPK Studies and Translational Medicine

BioDuro integrates various key disciplines required to achieve the program goals of development, including drug discovery, early-stage characterization, preformulation and formulation testing, in vitro testing, drug design and DMPK studies. Uniting the key disciplines involves integrating chemistry, physical sciences, computeraided drug discovery (CADD), biology, ADMET/PK and disease models.

Since the preclinical studies, formulation development, in vitro and animal tests are conducted in tandem by the same outsource partner along parallel paths, drug sponsors save considerable time — and consequently costs — by utilizing a single team of experts throughout development.

Another benefit of this integrated approach is that the early completion of characterization and other studies provides valuable information earlier, to guide formulation development and the final dosage form. As a result, companies can realize at an earlier stage whether the formulation will work or carries too much risk, which can save millions of dollars.

The burgeoning costs of healthcare globally are not sustainable. The Tufts Center for the Study of Drug Development reports that it costs on average US$2.6 million and 15 years to develop and obtain regulatory approval for a new drug. Driving up drug prices is the fact that only 12 per cent of drug candidates entering clinical trials result in an approved drug, and only 20 per cent of innovator companies recoup their investment. Ageing populations, with increased chronic healthcare needs, further add to healthcare costs. For example, more than one quarter of the Japanese population is older than 65 years of age, and this percentage is expected to rise to 45 per cent by 2050.1

To control these increasing costs, innovation and optimisation are required from all sectors of the healthcare ecosystem. This paper will show that by leveraging contemporary modelinformed drug development strategies, cost-effective medicines can be delivered to patients.

Inspiring Change

Certara believes in global equity in patient access to cost-effective medicines. To that end, the company strives to increase the efficiency of drug development through an integrated approach, informed by quantitative in silico models. These models inform clinical trial design and comparative effectiveness evaluation, optimise dosing, predict drug safety, and reduce the unnecessary exposure of patients and animals to experimental drugs in clinical trials. These methods have also driven new regulatory and drug pricing policies.Rate of Discovery of New Drugs Assignment Paper

Regulatory Acceptance

During the past three years, Certara technology and services supported 90 per cent of drugs approved by the US Food and Drug Administration (FDA). Further highlighting the importance of such quantitative methods, US FDA Commissioner Dr. Scott Gottlieb in 2017 stated that the FDA is using quantitative in silico tools as part of its comprehensive Innovation Initiative ‘to predict clinical outcomes, inform clinical trial designs, support evidence of effectiveness, optimise dosing, predict product safety, and evaluate potential adverse event mechanisms.’

Many other global regulatory agencies such as the European Medicines Agency (EMA), Australian Therapeutic Goods Administration (TGA), and Japanese Pharmaceuticals and Medical Devices Agency (PMDA) also embrace Model-Informed Drug Development (MIDD) and have issued guidances for its use to support regulatory filings.

Further underpinning the importance of MIDD, the PMDA announced that between 2014 and 2016, Physiologicallybased Pharmacokinetic (PBPK) modelling and simulation reports were included in 17 NDAs.2 In those instances, PBPK was used primarily to evaluate drug-drug interactions (DDIs), predict drug exposure in paediatric patients, and determine the impact of ethnic differences and disease states on drug PK.2

Guided by the target product profile and the goal of developing cost-effective drugs, Certara supports the drug sponsor’s R&D program by employing MIDD to translate animal study data to humans, improve clinical trial design, determine the optimal drug dose and dose regimen, assess safety and efficacy across the exposure range, evaluate the potential for DDIs, and inform drug labels. The key result is expected improvement in technical and commercial success.

MIDD Solutions

MIDD technologies include Pharmacokinetic (PK) and Pharmacokinetic / Pharmacodynamic (PK/PD) modelling tools; PBPK and Quantitative Systems Pharmacology (QSP) platforms; and emerging strategies such as Model-based Meta-Analysis (MBMA) And Pharmacology to Payer (P2P).

PBPK and QSP

While PK and population PK (PopPK) modelling are drug focused, PBPK provides mechanistic detail, and QSP is more disease mechanism focused.

PBPK informs drug dose, dosing regimens and DDIs. It is also used for bridging studies to establish safety and efficacy in special populations, including paediatrics, ethnic, the elderly, pregnant women, and patients with renal or hepatic impairment.

QSP integrates quantitative drug data with systems biology knowledge, such as mechanisms of action. US FDA is interested in QSP for its ability to address the high rates of drug failure by providing the missing link between target modulation and clinical efficacy / safety outcomes. Certara routinely applies QSP in the lead optimisation phase to help determine which assets to progress.Rate of Discovery of New Drugs Assignment Paper

MBMA examines curated publicly-available data and proprietary clinical data for drugs in development or on market for a particular clinical indication and predicts the probability of technical and commercial success for a proprietary drug. MBMA evaluates comparative effectiveness, safety and tolerability. It is also used to test target product profiles, optimise clinical trial designs, and provide decision support for portfolio and marketing strategy.

In parallel, the company provides due diligence services for investors, and pharma licensing evaluations. It also develops regulatory strategy, participates in regulatory meetings, and prepares new product filings for global regulatory agencies.

Communicating Drug Cost-benefit to Different Stakeholders

Regulatory approval alone does not guarantee drug reimbursement and patient access to new drugs. Payers and health authorities must be convinced of a new drug’s value for it to be placed on the formulary, factored into reimbursement rates, incorporated into treatment plans and prescribed to patients. Historically, discussions with payers only occurred in late clinical development. To facilitate earlier payer engagement, we have developed quantitative frameworks to provide value-based narratives for engaging stakeholders regarding the features and benefits of experimental drugs. By integrating HEOR and real-world value assessments with pharmacometrics data, we can deliver safety, efficacy and effectiveness insights from as early as Phase 1 clinical trials, through the product life cycle, and into health technology assessment and payer decisions.

Certara has called one of these ‘end to end’ frameworks, Pharmacology to the Payer (P2P)3. The P2P framework provides an agreed lexicon between stakeholders which integrate data into an agent-based model incorporating pharmacology, epidemiology, and health economics for a given drug. The P2P framework has major applications for preparedness, planning and deployment of medical countermeasures for infectious diseases.

The following two examples show how MIDD can solve different drug development challenges.Rate of Discovery of New Drugs Assignment Paper

Oncology Case Study

Mantle Cell Lymphoma (MCL) is a rare form of non-Hodgkin lymphoma that affects about 3,000 patients per year in the US4. When Imbruvica, a tyrosine kinase inhibitor, was shown to be an effective treatment for MCL, it was submitted to the US FDA’s Accelerated Approval Program. That application was successful and Imbruvica became one of the first drugs to be awarded breakthrough status by the US FDA.

Due to the known involvement of CYP3A in the metabolism of Imbruvica, it was necessary to identify potential DDIs in different patient populations and to define the dose and dosing regimen in those populations.

Certara’s Simcyp® PBPK Simulator was used to gain a better understanding of Imbruvica’s PK profile and evaluate its DDI liability. The Imbruvica PBPK model was developed using in vitro and clinical DDI data, validated using known inhibitors and inducers of CYP3A, and applied to evaluate untested clinical DDI scenarios. This PBPK model informed the Imbruvica label, providing guidance for clinicians on 24 untested DDI scenarios, and a dose optimisation strategy for individuals.

Imbruvica is now approved for MCL, first line treatment for chronic lymphocytic leukaemia, and for the treatment of adult patients with chronic graft-versushost-disease that is not responding to other standard therapies.

The US FDA highlighted this Imbruvica case study during one of its workshops as an example of a successful application of PBPK predictions to fill in clinical gaps during the evaluation of a breakthrough drug treatment. PBPK is a key tool in the MIDD armamentarium informing precision dosing in different patient sub-populations.

Respiratory Syncytial Virus (RSV) Case Study

In this case, PopPK and PK/PD, together with viral kinetic models and contemporary clinical trial design, delivered high value in an RSV early development program. RSV often infects paediatric patients, the majority of which are under two years of age. While the morbidity and mortality are high in this patient population, there is no effective treatment and no precedent for regulatory acceptance of an accelerated development pathway for RSV therapeutics in infants.

Developing RSV medicines for paediatric patients is both scientifically and operationally challenging. As RSV infection is a seasonal disease, it is difficult to identify paediatric patients and execute clinical trials efficiently. Furthermore, some parents are reluctant to give consent for their infant to be included in such clinical trials.

ALS-8176, an anti-RSV compound, was being developed for paediatric patients. Its safety and PK had been characterised in preclinical disease models and clinical trials in healthy adults. The challenge was how to move from trials in healthy adults to infants under two years of age in a timely manner.Rate of Discovery of New Drugs Assignment Paper

The development team considered a Human Challenge Model (HCM) — where healthy adults are infected with RSV and then treated with the drug — to be the most relevant translational medicine tool for bridging to children.

The development team was confident that the PK/PD determinants of ALS-8176 efficacy could be established in an HCM. These PK/PD readouts inform ‘therapeutic exposures,’ which PopPK and PBPK models then assist in converting from adult to paediatric doses to be evaluated in future paediatric clinical trials. This type of translational medicine and MIDD bridging program also makes it possible to accelerate drug development for paediatric populations.

The cost and time required to conduct the HCM study were significant considerations for the biotech team. Therefore, it agreed to embark on a novel adaptive design for its HCM clinical trial, which used the evolving PK/PD data in real time to develop a picture of the underlying exposure response relationship for ALS-8176.

Evolving clinical trial data for ALS-8176 were used to augment the MIDD model and predict subsequent cohort dosing decisions. Those dosing decisions included whether to increase or decrease the next dose, incorporate a loading dose regimen, recruit additional subjects at a given dose, and when to stop the clinical trial. This “learn-confirm” approach was continued during the clinical trial between cohorts, successfully identifying the PK/PD targets to inform the ongoing program. In this way, the exposure response surface was very efficiently established.

In this example, rich PK and PD data from the HCM were coupled with PopPK and PBPK, to deliver robust recommendations on dosing regimens to be studied in RSV-infected infants under two years of age.

The adaptive design of the HCM study required only about 50 per cent of the patients needed for a conventional placebo-controlled trial, saving an estimated six months of development time and more than five million dollars. The study was published in the New England Journal of Medicine.5

Furthermore, this integrated MIDD program set new precedent when EMA accepted this scientific strategy in support of an accelerated development pathway to paediatrics. It permitted the movement of ALS-8176 from healthy adults to RSV-infected children under the age of two very early in the drug development process.

Both case studies showcase compounds that helped strengthen the sponsor’s portfolio, whereby these small biotechs were acquired at a significant valuation by leading pharmaceutical companies.

These cases demonstrate the many ways in which MIDD adds value to a drug development program. It can provide strong scientific evidence to improve decision making; support leaner study designs and reduce time and costs; develop new regulatory pathways; and facilitate regulatory science innovations and ecosystem improvements. It can also demonstrate drug value to payers and help expedite getting new medicines to the patients that need them.Rate of Discovery of New Drugs Assignment Paper

Developing a new drug takes years of research and testing before it gets approved by FDA. The procedure of FDA approval is lengthy too and it takes many years before an approved drug hits the market. When it comes to developing a new drug, the factors pertaining uncertainty are high. Drug development is a challenging phase, especially, when new drugs for genetic or chronic ailments are introduced for research and development.

 

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Identifying the Class of Drugs

This is an important aspect of drug development. The age and other conditions are analyzed before producing a formulation for a new drug. These formulations must prove to be effective at the cellular, biochemical, and functional level for the human body. During the process of development, the number of compounds to produce the formulation is reduced to two to three chemicals at the most.

 

Clinical Trials and Regulations
Clinical trials are the lengthiest procedures used for a new drug
There are also investigations conducted at each and every stage of drug development
A new drug is tested on a human subject with the availability of doctors and healthcare professionals
The reaction for the active and inactive chemicals in the compounds is tested
Its effectiveness is analyzed through behavioral and physical changes in the patient
There are different phases of conducting clinical trials

Drug Safety for Public Use

Only healthy volunteers are tested on new drugs. The absorption rate and its effectiveness are tested till the point of excretion of the drug. The treatment for which the drug is introduced is checked for its effect as a remedy to that ailment. At the next stage, its effect on different population with different quantity of dosage is checked and analyzed.

The Investigational New Drug Process    

Drug developers, or sponsors, must submit an Investigational New Drug (IND) application to FDA before beginning clinical research.

In the Investigational New Drug IND application, developers must include:

Animal study data and toxicity (side effects that cause great harm) data
Manufacturing information
Clinical protocols (study plans) for studies to be conducted
Data from any prior human research
Information about the investigator Rate of Discovery of New Drugs Assignment Paper

The FDA authorities visit laboratories, research and development centers, as well as manufacturing units to check for the safety of the drugs being developed. The investment made in drug development gives unpredictable results.

Drug development process

A variety of approaches is employed to identify chemical compoundsthat may be developed and marketed. The current state of the chemical and biological sciences required for pharmaceuticaldevelopment dictates that 5,000–10,000 chemical compounds must undergo laboratory screening for each new drug approved for use in humans. Of the 5,000–10,000 compounds that are screened, approximately 250 will enter preclinical testing, and 5 will enter clinical testing. The overall process from discovery to marketing of a drug can take 10 to 15 years. This section describes some of the processes used by the industry to discover and develop new drugs. The flowchart provides an overall summary of this developmental process

Research and discovery
Pharmaceuticals are produced as a result of activities carried out by a complex array of public and private organizations that are engaged in the development and manufacture of drugs. As part of this process, scientists at many publicly funded institutions carry out basic research in subjects such as chemistry, biochemistry, physiology, microbiology, and pharmacology. Basic research is almost always directed at developing new understanding of natural substances or physiological processes rather than being directed specifically at development of a product or invention. This enables scientists at public institutions and in private industry to apply new knowledge to the development of new products. The first steps in this process are carried out largely by basic scientists and physicians working in a variety of research institutions and universities. The results of their studies are published in scientific and medical journals. These results facilitate the identification of potential new targets for drug discovery. The targets could be a drug receptor, an enzyme, a biological transport process, or any other process involved in body metabolism. Once a target is identified, the bulk of the remaining work involved in discovery and development of a drug is carried out or directed by pharmaceutical companies.Rate of Discovery of New Drugs Assignment Paper

Contribution of scientific knowledge to drug discovery
Two classes of antihypertensive drugs serve as an example of how enhanced biochemical and physiological knowledge of one body system contributed to drug development. Hypertension (high blood pressure) is a major risk factor for development of cardiovascular diseases. An important way to prevent cardiovascular diseases is to control high blood pressure. One of the physiological systems involved in blood pressure control is the renin-angiotensin system. Renin is an enzyme produced in the kidney. It acts on a blood protein to produce angiotensin. The details of the biochemistry and physiology of this system were worked out by biomedical scientists working at hospitals, universities, and government research laboratories around the world. Two important steps in production of the physiological effect of the renin-angiotensin system are the conversion of inactive angiotensin I to active angiotensin II by angiotensin-converting enzyme (ACE) and the interaction of angiotensin II with its physiologic receptors, including AT1 receptors. Angiotensin II interacts with AT1 receptors to raise blood pressure. Knowledge of the biochemistry and physiology of this system suggested to scientists that new drugs could be developed to lower abnormally high blood pressure.

A drug that inhibited ACE would decrease the formation of angiotensin II. Decreasing angiotensin II formation would, in turn, result in decreased activation of AT1 receptors. Thus, it was assumed that drugs that inhibit ACE would lower blood pressure. This assumption turned out to be correct, and a class of antihypertensive drugs called ACE inhibitors was developed. Similarly, once the role of AT1 receptors in blood pressure maintenance was understood, it was assumed that drugs that could block AT1 receptors would produce antihypertensive effects. Once again, this assumption proved correct, and a second class of antihypertensive drugs, the AT1 receptor antagonists, was developed. Agonists are drugs or naturally occurring substances that activate physiologic receptors, whereas antagonistsare drugs that block those receptors. In this case, angiotensin II is an agonist at AT1 receptors, and the antihypertensive AT1 drugs are antagonists. Antihypertensives illustrate the value of discovering novel drug targets that are useful for large-scale screening tests to identify lead chemicals for drug development.Rate of Discovery of New Drugs Assignment Paper

Drug screening

Sources of compounds
Screening chemical compounds for potential pharmacological effects is a very important process for drug discovery and development. Virtually every chemical and pharmaceutical company in the world has a library of chemical compounds that have been synthesized over many decades. Historically, many diverse chemicals have been derived from natural products such as plants, animals, and microorganisms. Many more chemical compounds are available from university chemists. Additionally, automated, high-output, combinatorial chemistry methods have added hundreds of thousands of new compounds. Whether any of these millions of compounds have the characteristics that will allow them to become drugs remains to be discovered through rapid, high-efficiency drug screening

Lead chemical identification

It took Paul Ehrlich years to screen the 606 chemicals that resulted in the development of arsphenamine as the first effective drug treatment for syphilis. From about the time of Ehrlich’s success (1910) until the latter half of the 20th century, most screening tests for potential new drugs relied almost exclusively on screens in whole animals such as rats and mice. Ehrlich screened his compounds in mice with syphilis, and his procedures proved to be much more efficient than those of his contemporaries. Since the latter part of the 20th century, automated in vitro screening techniques have allowed tens of thousands of chemical compounds to be screened for efficacyin a single day. In large-capacity in vitro screens, individual chemicals are mixed with drug targets in small, test-tube-like wells of microtiter plates, and desirable interactions of the chemicals with the drug targets are identified by a variety of chemical techniques. The drug targets in the screens can be cell-free (enzyme, drug receptor, biological transporter, or ion channel), or they can contain culturedbacteria, yeasts, or mammalian cells. Chemicals that interact with drug targets in desirable ways become known as leads and are subjected to further developmental tests. Also, additional chemicals with slightly altered structures may be synthesized if the lead compound does not appear to be ideal. Once a lead chemical is identified, it will undergo several years of animal studies in pharmacology and toxicology to predict future human safety and efficacy.Rate of Discovery of New Drugs Assignment Paper

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Lead compounds from natural products

Another very important way to find new drugs is to isolate chemicals from natural products. Digitalis, ephedrine, atropine, quinine, colchicine, and cocaine were purified from plants. Thyroid hormone, cortisol, and insulin originally were isolated from animals, whereas penicillin and other antibiotics were derived from microbes. In many cases plant-derived products were used for hundreds or thousands of years by indigenous peoples from around the world prior to their “discovery” by scientists from industrialized countries. In most cases these indigenous peoples learned which plants had medicinal value the same way they learned which plants were safe to eat—trial and error. Ethnopharmacology is a branch of medical science in which the medicinal products used by isolated or primitive people are investigated using modern scientific techniques. In some cases chemicals with desirable pharmacological properties are isolated and eventually become drugs with properties recognizable in the natural product. In other cases chemicals with unique or unusual chemical structures are identified in the natural product. These new chemical structures are then subjected to drug screens to determine if they have potential pharmacological or medicinal value. There are many cases where such chemical structures and their synthetic analogs are developed as drugs with uses unlike those of the natural product. One such compound is the important anticancer drug taxol, which was isolated from the Pacific yew (Taxus brevifolia).

Taxol and the Pacific yew
As a member of the yew family, Taxaceae, the Pacific yew (Taxus brevifolia) has flat, evergreen needles and produces red, berrylike fruits. The toxicity of members of the yew family was described in ancient Greek literature. Indeed, the genus name Taxus derives from the Greek word toxon, which can be translated as toxin or poison. Pliny the Elder described people who died after drinking wine that had been stored in containers made from yew wood. Julius Caesardescribed how one of his enemies, Catuvolcus, poisoned himself using a yew plant. The early Japanese used yew plant parts to induce abortion and to treat diabetes, and Native Americans used yew to treat arthritis and fever. In part because of widespread historical accounts of the pronounced biological effects inherent in members of the yew family, samples of the Pacific yew were included in screens for potential anticancer drugs.Rate of Discovery of New Drugs Assignment Paper

This screening process was initiated as a cooperative venture between the United States Department of Agriculture (USDA) and the National Cancer Institute (NCI) of the United States. Extracts from the Pacific yew were tested against two cancer cell lines in 1964 and found to have promising effects. After a sufficient quantity of the extract was prepared, the active compound, taxol, was isolated in 1969. In 1979 pharmacologist Susan Horwitz and her coworkers at Yeshiva University’s Albert Einstein College of Medicine reported a unique mechanism of action for taxol. In 1983 NCI-supported clinical trials with taxol were begun, and by 1989 NCI-supported clinical researchers at Johns Hopkins University reported very positive effects in the treatment of ovarian cancer. Also in 1989 the NCI reached an agreement with Bristol-Myers Squibb to increase production, supplies, and marketing of taxol. Taxol marketing for the treatment of ovarian cancer began in 1992. Bristol-Myers Squibb applied to trademark the name taxol, which became Taxol®, and the generic name became paclitaxel.

Initially, the sole source of taxol was the bark of the Pacific yew, native to the old-growth forests along the northwest coast of the United States and in British Columbia. This led to considerable public controversy. Environmental groups feared that harvesting of the yew would endanger its survival. It took the bark of between three and ten 100-year-old plants to make enough drug to treat one patient. There were also fears that harvesting the yew would lead to environmental damage to the area and could potentially destroy much of the habitat for the endangered spotted owl. After several years of controversy, Bristol-Myers Squibb adopted a semisynthetic process for making taxol. This process uses a precursor, which is chemically converted to taxol. The precursor is extracted from the needles (renewable biomass) of Taxus baccata, which is grown in the Himalayas and in Europe. Although there were some political controversies surrounding the discovery and development of taxol, the story of its development and marketing provides another example of how public and private enterprise can cooperate in the development of new discoveries and new drugs.

Strategies for drug design and production

The term structure-activity relationship (SAR) is now used to describe the process used by Ehrlich to develop arsphenamine, the first successful treatment for syphilis. In essence, Ehrlich synthesized a series of structurally related chemical compounds and tested each one to determine its pharmacological activity. In subsequent years many drugs were developed using the SAR approach. For example, the β-adrenergic antagonists (antihypertensive drugs) and the β2agonists (asthma drugs) were developed initially by making minor modifications to the chemical structure of the naturally occurring agonists epinephrine (adrenaline) and norepinephrine (noradrenaline). Once a series of chemical compounds had been synthesized and tested, medicinal chemists began to understand which chemical substitutions would produce agonists and which would produce antagonists. Additionally, substitutions that would cause metabolic enzyme blockade and increase the gastrointestinal absorption or duration of action began to be understood. Three-dimensional molecular models of agonists and antagonists that fit the drug receptor allowed scientists to gain important information about the three-dimensional structure of the drug receptor site. By the 1960s SAR had been further refined by creating mathematical relationships between chemical structure and biological activity. This refinement, which became known as quantitative structure-activity relationship, simplified the search for chemical structures that could activate or block various drug receptors.Rate of Discovery of New Drugs Assignment Paper

Computer-aided design of drugs
A further refinement of new drug design and production was provided by the process of computer-aided design (CAD). With the availability of powerful computers and sophisticated graphics software, it is possible for the medicinal chemist to design new molecules and evaluate their potential interaction with a receptor or an enzyme before they are synthesized. This means that the chemist may be able to synthesize and test only the most promising compounds, thus allowing potential new drugs to be synthesized more efficiently and cheaply.

Combinatorial chemistry
Combinatorial chemistry was a development of the 1990s. It originated in the field of peptide chemistry but has since become an important tool of the medicinal chemist. Traditional organic synthesis is essentially a linear process with molecular building blocks being assembled in a series of individual steps. Part A of the new molecule is joined to part B to form part AB. After part AB is made, part C can be joined to it to make ABC. This step-wise construction is continued until the new molecule is complete. Using this approach, a medicinal chemist can, on average, synthesize about 25 new compounds per year. In combinatorial chemistry, one might start with five compounds (A1–A5). These five compounds would be reacted with building blocks B1–B5 and building blocks C1–C5. These reactions take place in parallel rather than in series, so that A1 would combine with B1, B2, B3, B4, and B5. Each one of these combinations would also combine with each of the C1–C5 building blocks, so that 125 compounds would be synthesized. Using robotic synthesis and combinatorial chemistry, hundreds of thousands of compounds can be synthesized in much less time than would have been required to synthesize a few compounds in the past.Rate of Discovery of New Drugs Assignment Paper

Synthetic human proteins
Another important milestone for medical science and for the pharmaceutical industry occurred in 1982, when regulatory and marketing approval for Humulin®, human insulin, was granted in the United Kingdom and the United States. This marketing approval was an important advancement because it represented the first time a clinically important, synthetic human protein had been made into a pharmaceutical product. Again, the venture was successful because of cooperative efforts between physicians and scientists working in research institutions, universities, hospitals, and the pharmaceutical industry.

Human insulin is a small protein composed of 51 amino acids and has a molecular weight of 5,808 daltons (units of atomic mass). The amino acid sequence and chemical structure of insulin had been known for a number of years prior to the marketing of Humulin®. Indeed, the synthesis of sheep insulin had been reported in 1963 and human insulin in 1966. It took almost another 20 years to bring synthetic human insulin to market because a synthetic process capable of producing the quantities necessary to supply market needs had not been developed.Rate of Discovery of New Drugs Assignment Paper

In 1976 a new pharmaceutical firm, Genentech Inc., was formed. The goal of Genentech’s founders was to use recombinant DNA technology in bacterial cells to produce human proteins such as insulin and growth hormone. Since the amino acid sequence and chemical structure of human insulin were known, the sequence of DNA that coded for synthesis of insulin could be reproduced in the laboratory. The DNA sequence coding for insulin production was synthesized and incorporated into a laboratory strain of the bacteria Escherichia coli. In other words, genes made in a laboratory were designed to direct the synthesis of insulin in bacteria. Once the laboratory synthesis of insulin by bacteria was completed, scientists at Genentech worked with their counterparts at Eli Lilly & Co. to scale up the new synthetic process so that marketable quantities of human insulin could be made. Regulatory approval for marketing human insulin came just six years after Genentech was founded.

In some ways, the production of human growth hormone by recombinant DNA technology, first approved for use in 1985, was more important than the synthesis of insulin. Prior to the availability of human insulin, most people with diabetes could be treated with the bovine or porcine insulin products, which had been available for 50 years (see above Isolation of insulin). Unlike insulin, the effects imparted by growth hormone are different for every species. Therefore, prior to the synthesis of human growth hormone, the only source of the human hormone was from cadaver pituitaries. However, there are now a number of recombinant preparations of human growth hormone and other human peptides and proteins on the market.Rate of Discovery of New Drugs Assignment Paper

Drug Regulation And Approval
Regulation by government agencies
Concerns related to the efficacy and safety of drugs have caused most governments to develop regulatory agencies to oversee development and marketing of drug products and medical devices. Use of any drug carries with it some degree of risk of an adverse event. For most drugs the risk-to-benefit ratio is favourable; that is, the benefit derived from using the drug far outweighs the risk incurred from its use. However, there have been unfortunate circumstances in which drugs have caused considerable harm. The harm has come from drug products containing toxic impurities, from drugs with unrecognized severe adverse reactions, from adulterated drug products, and from fake or counterfeit drugs. Because of these issues, effective drug regulation is required to ensure the safety and efficacy of drugs for the general public.

Public influence on drug regulation
The process of drug regulation has evolved over time. Laws regulating drug marketing and development, government regulatory agencies with oversight of drug development and use, drug evaluation boards, drug information centres, and quality control laboratories have become part of the cooperative venture that produces and develops drugs. In some countries drug laws omit or exempt certain areas of pharmaceutical activity from regulation. For example, some countries exempt herbal or homeopathic products from regulation. In other countries there is very little regulation imposed on drug importation. Over time, the scope of drug laws and the authority vested in regulatory agencies have gradually expanded. In some instances, strengthening of drug laws has been the result of a drug-related catastrophe that prompted public demand for more restrictive legislation to provide more protection for the public. One such example occurred in the 1960s with thalidomide that was prescribed to treat morning sickness in pregnant women. Thalidomide had been on the market for several years before it was realized to be the causative agent of a rare birth defect, known as phocomelia, that had begun appearing at epidemic proportions. There was a dramatic reaction to the devastation caused by thalidomide, especially because it was considered a needless drug.Rate of Discovery of New Drugs Assignment Paper

At other times the public has perceived that drug regulation and regulatory authorities have been too restrictive or too cautious in approving drugs for the market. This concern typically has been related to individuals with serious or life-threatening illnesses who might benefit from drugs that have been denied market approval or whose approval has been inordinately delayed because regulations are too strict. At times, governments have responded to these concerns by streamlining drug laws and regulations. Examples of types of drugs given expedited approval are cancer drugs and AIDSdrugs. Regulatory measures that make rapid approval of new drugs paramount sometimes have led to marketing of drugs with more toxicity than the public finds acceptable. Thus, drug regulations can and probably will remain in a state of flux, becoming more lax when the public perceives a need for new drugs and more strict following a drug catastrophe.

The Importance of the Team in Drug Development

 

In recent years, many pharmaceutical development experts have published opinion pieces focused on the issues faced by their industry. There are calls for changes in drug design workflows, and many focus on the need for inter-disciplinary research teams that share data and embrace modern research solutions.

One such expert is Dr. Scott Lusher, former Director of Strategy & Communications at the Netherlands eScience Center, an organization that supports the development of new IT solutions that better reflect and leverage the new, data-laden nature of scientific research. In aninterview with Elsevier, he detailed how big data could only benefit drug design and optimization if there was a significant change in the approach to pharmaceutical development.

In the fascinating interview (which I encourage you to read for yourself), Dr. Lusher shared his opinions on the questions that should drive the drug design cycle, how data should be handled, and what technology could best facilitate the recording, analysis, sharing and visualization of information.

I found his opinions about teamwork particularly interesting. He pointed out that all the features and analysis power of technology will always remain secondary to the interpretation of data: that is a role that will still be done by people. And in order to work best, he says that a change in mentality is required:

“Medicinal chemists, biologists, pharmacologists and all other team members will need to work with, share, trust and interpret data differently. Currently, each team member has a specific task: the medicinal chemists do the design and synthesis, the biologists perform bioassays, the toxicologists search for potential adverse effects, and so on. Those boundaries will need to vanish. While each individual brings his or her expertise to the group, all must be versed in every discipline encompassed by the team and all must work at the interface of these fields.”

Dr. Lusher goes on to say that the pharmaceutical industry needs to reassert the importance of the team, as he believes that “concrete decisions about the direction of a compound series are best made by all hands—the team of people that generated and know their data.”Rate of Discovery of New Drugs Assignment Paper

In corporate, customer-focused settings, inter-functional teams are a common approach to complex projects, with the mix of skills and knowledge recognized as necessary for success. Dr. Lusher is recommending that these teams elevate to a new level of collaboration where individual members become active participants in every aspect of the drug development workflow. Team members must look beyond the boundaries of their own tasks and fully understand the work and results of every development step to transform the team into a synergistic unit that can make informed decisions based on the whole of data they produce. It would be fascinating to know if any companies are already applying this type of teamwork and what the impact has been.

Non-clinical / Pre-clinical Development

In non-clinical development you must always expect the unexpected!

Non-clinical testing is conducted throughout all phases of drug development in order to assess the safety profile and pharmacokinetic and toxicokinetic (PK/TK) characteristics of candidate medicinal products. If the non-clinical (or preclinical) development is performed well, it can maximize the chances of success in the clinical development phases. Strategies for the non-clinical development of products follow general regulatory guidelines, but are also designed on a case-by-case basis according to the specifics of the drug. It is of key importance to design an optimal preclinical development program that allows the drug to be taken forward into the clinic (“e.g. IND package”), or a more extended non-clinical development plan allowing the drug to be taken into the next clinical phase, or to product registration (e.g. including 6 months tox, carcinogenicity study, reproductive toxicity etc). The project managers of Venn can guide the client though all these phases of drug development.

The non-clinical experts of Venn have a broad experience and hands-on drug development expertise with respect to design, monitoring, issue management and overall consultancy for PK/TK, metabolism, safety pharmacology and toxicology studies. Questions that are handled by the Venn expert range from writing responses to questions obtained from authorities (FDA/EMA) to calculating a safe starting dose for a first-in-man study (NOAEL/MABEL) and from advising on specific study findings (e.g QTc prolongation, or unexpected deaths in a study) to proposing a strategy on how to deal with high levels of metabolites (MIST guideline). Also for obtaining a second opinion on any pre-clinical or non-clinical research issue/question, the Venn experts can be consulted.

The non-clinical experts work in close collaboration with the Venn project managers CMC, regulatory, QA and clinical specialists to ensure that non-clinical (or preclinical) development plans provide the most time-efficient and cost-effective strategy for clients.Rate of Discovery of New Drugs Assignment Paper

Pharmacokinetics and Toxicokinetics (PK/TK)
Non-clinical / Preclinical Project Management
Preparation of Non-clinical Regulatory Documentation and Reports
Non-clinical (Preclinical) Consultancy

1. Introduction

Microbes play an essential role in the production of antibiotics, antifungal as well as antiviral infections and this role is expanding each day (1). Owing to their ability to produce useful secondary metabolites, microbes have contributed greatly in the development of pharmaceutical industry and the control of many medical conditions as they are now widely used as antitumour drugs, immunosuppressants, enzyme inhibitors, and in many other applications (1). Back in 1928, Alexander Fleming found a compound produced by a mould, which was later identified as Penicillium notatum, had the capability of killing the bacterium Staphylococcus aureus in his laboratory. The active compound was known as penicillin, a beta-lactam antibiotic, and it was used massively as a potent antimicrobial drug during World War II (1). This discovery has marked the beginning of the microbial drug era as many useful antibiotics have since been isolated from soil bacteria, for instance, streptomycin, chloramphenicol, and tetracycline (2). These antibiotics produced have had remarkable biological activities to human beings, to illustrate, streptomycin was the first active drug against tuberculosis whereas chloramphenicol was the drug of choice for typhoid fever (3-5).

The soil is incontestably a rich reservoir that allows the screening of drug compounds as it can harbour an enormous number of soil inhabitants, such as bacteria, fungi, algae, protozoa, insects, and other more complex living organisms (6). Many soil organisms have the ability to produce secondary metabolites which enable them to inhibit the growth of other microorganisms in the same niche in order to compete for survival (7). Over the past decades, a significant amount of bioactive compounds have been discovered from the terrestrial environment (3). The majority of the useful soil microorganisms belong to the genera Penicillium, Streptomyces, Cephalosporium, Micromonospora and Bacillus, and they are still being studied continuously (8). Bacillus is found abundantly in the soil environment and it is known to produce antibiotic like bacitracin, pumulin and gramicidin which are active against Gram-positive bacteria, and polymyxin, colistin and circulin which are effective against Gram-negative bacteria, demonstrating a vast range of antimicrobial activity (9; 10). Furthermore, more than 60% of antimicrobial agents used in human and animals were originated from the genus Streptomyces, some of which are chloramphenicol, erythromycin, and tetracycline, which have a broad spectrum of activity (5; 9).Rate of Discovery of New Drugs Assignment Paper

In the recent years, the search for antibiotics has plateaued whereby limited new antibacterial drugs are being introduced to the market, posing a challenge to the healthcare sector. The rate of discovery of new compounds has declined and the scientists are facing a bottleneck where the same known antimicrobial compounds have been isolated over the past few years (11). Nowadays, antibiotics are widely used therapeutically and prophylactically in the healthcare setting for human, animals and agricultural purposes. They are commercially exploited and the overconsumption of these compounds has brought about a rapid evolution in microorganisms where resistance to the antibiotics is spreading dramatically. Furthermore, the crisis of multidrug resistance is expanding uncontrollably, leading to a consistent demand for more effective and useful antibacterial medicines. Staphylococcus aureus, Pseudomonas sp., Klebsiella sp., and Enterococcus sp. are examples of common nosocomial bacteria and they often cause a serious problem to hospitalised patients (1). The development of resistance of these pathogens to currently available antibiotics, both natural and synthetic classes of antibiotics, has rendered many of the standard drugs ineffective (12).

New medicines are constantly under development. However, the Food and Drug Administration (FDA) has strict safety protocols on the steps new drugs must go through before people can use them. Given that approximately 90 percent of pharmaceuticals fail their clinical trials despite passing animal studies, and there are thousands of chemicals whose effects on the human body are unknown, the ability to easily distinguish the harmful from the helpful would be extremely beneficial.

Acute toxicity studies: Involve single-dose studies in animals are an important first step in establishing a safety profile, with the general aim of exploring a feasible dose range.Rate of Discovery of New Drugs Assignment Paper

Repeated-dose toxicity studies: The studies are designed to identify safe levels of the drug following treatment regimens that are designed to provide continuous exposure of the animals to the test drug. Ideally, the route of administration should be the same as that planned in humans, whereas the animal studies should involve higher doses and longer durations of exposure than those planned clinically. Toxicokinetic data are also obtained from repeated-dose toxicity studies and generally determine the plasma concentrations of the drug and are generally collected on the first day of dosing and near the last day.

Subchronic/Chronic Toxicity Studies: The maximum duration of chronic studies is generally 6 months, although the ICH guidelines describe situations where studies of 9–12 month duration may be necessary in a non rodent species. The usual in life and postmortem observations are also performed.

Pharmacokinetic studies: In the early stages of drug development, it is important to identify important parameters that relate to the absorption and excretion pathways for the drug. In the later stages of development, studies on the extent of tissue distribution and the identification of metabolites become important. In some situations, where single-dose tissue distribution studies suggest drug localization, a tissue distribution study following repeated dosing may be indicated. The conditions under which such studies may be necessary have been delineated in an ICH guideline (Federal Register, 1 March 1997).

Reproductive toxicity: Studies for reproductive effects examine the possibility that agents may affect fertility of male or female, by specific pharmacological or biochemical means or by toxicity to a number of cell types, including gametes and their supporting cells. Some agents may alter the delicate hormone balance required for the mammalian reproductive process to maintain its cyclical progress.Rate of Discovery of New Drugs Assignment Paper

Developmental toxicity: This concentrates on the most sensitive part of gestation, from the time of implantation until major organogenesis is complete. This is the period during which a test substance is most likely to cause malformation of the embryo.

Juvenile toxicity studies: Juvenile toxicity studies are recommended by both the Japanese and US regulatory agencies before inclusion of children in clinical trials. The studies are usually conducted in the offspring of untreated female rats by giving test material directly to the pups. Dosing usually does not commence until 4 days post partum, and is continued for 6 weeks.

Genotoxicity / Mutagenicity: These are in vivo or in vitro tests, conducted to detect compounds which induce genetic damage directly, or indirectly. These tests enable a hazard identification, with respect to damage to DNA, and its fixation.

Carcinogenicity studies: Carcinogenicity studies involve the treatment of rodents for long periods, approximating to the complete life span, in order to determine whether the test material assesses the capability to initiate or promote the development of tumors. Carcinogenicity studies have been required for all drugs where clinical therapy may extend for six months or longer.

Thus preclinical studies are helpful in discovering possible hazards to human beings who are exposed to much lower doses compared to administration of high animal doses, and the data obtained helps in assessment of the relevance of these findings to clinical safety, to support the choice of species and treatment regimen in non-clinical toxicity studies. Animal toxicity study data are required for clinical trials to be carried out and marketing of new drugs. Preclinical data is helpful in increasing the overall strategic success.

Before testing a drug in people, researchers must find out whether it has the potential to cause serious harm, also called toxicity. The two types of preclinical research are:FDA requires researchers to use good laboratory practices (GLP), defined in medical product development regulations, for preclinical laboratory studies.  The GLP regulations are found in 21 CFR Part 58.1: Good Laboratory Practice for Nonclinical Laboratory Studies. These regulations set the minimum basic requirements for:

study conduct
personnel
facilities
equipment
written protocols
operating procedures
study reports
and a system of quality assurance oversight for each study to help assure the safety of FDA-regulated product

Usually, preclinical studies are not very large. However, these studies must provide detailed information on dosing and toxicity levels. After preclinical testing, researchers review their findings and decide whether the drug should be tested in people.

Before testing a drug in people, researchers must find out whether it has the potential to cause serious harm, also called toxicity. The two types of preclinical research are:

In Vitro
In Vivo

FDA requires researchers to use good laboratory practices (GLP), defined in medical product development regulations, for preclinical laboratory studies.  The GLP regulations are found in 21 CFR Part 58.1: Good Laboratory Practice for Nonclinical Laboratory Studies. These regulations set the minimum basic requirements for:

study conduct
personnel
facilities
equipment
written protocols
operating procedures
study reports
and a system of quality assurance oversight for each study to help assure the safety of FDA-regulated product

Usually, preclinical studies are not very large. However, these studies must provide detailed information on dosing and toxicity levels. After preclinical testing, researchers review their findings and decide whether the drug should be tested in people.Rate of Discovery of New Drugs Assignment Paper

Clinical trial phases
The entire process of moving a drug from design to clinical trials takes 10 to 12 years on average. Let’s take a closer look at each stage to better understand what goes into early clinical development and preparation for approval of a drug.
Preclinical studies
Deciding whether a drug is ready for clinical trials (the so-called move from bench to bedside) involves extensive preclinical studies that yield preliminary efficacy, toxicity, pharmacokinetic and safety information. Wide doses of the drug are tested using in vitro (test tube or cell culture) and in vivo (animal) experiments, and it is also possible to perform in silico profiling using computer models of the drug–target interactions.
Much like for clinical trials, there are certain types of trials that have to be done, such as toxicology studies in most cases, and other trials that are specific to the particular study compound or question. Understanding that the goal of preclinical trials is to move into the clinical stage is key and the studies should be designed around that goal. Watch our webinar on moving from preclinical to clinical trials.
Don’t get too worked up on too many preclinical trials that may not be necessary but make sure to consult with experts who can help you decide, which trials you should do and if you are ready to move into clinical stage. At Profil we have a team of experts who can advice you on such questions and who will help you with the transition into clinical trials. Contact us to start the discussion.
Phase 0 clinical trial
Phase 0 involves exploratory, first-in-human (FIH) trials that are run according to FDA guidelines. Also called human microdose studies, they have single sub-therapeutic doses given to 10 to 15 subjects and yield pharmacokinetic data or help with imaging specific targets without introducing pharmacological effects. Pharmaceutical companies perform Phase 0 studies to decide which of their drug candidates has the best pharmacokinetic parameters in humans.Rate of Discovery of New Drugs Assignment Paper

Phase I–IV versus early and late phase clinical trials

Clinical trials involving new drugs are commonly classified into four phases. However, they can also be classified as early phase clinical trials and late phase trials because there can be overlap between phases. Profil focuses on Phase I+II clinical trials as we are a full-service CRO for early clinical development.

Phase I

Phase I trials are the first tests of a drug with a small number of healthy human subjects. Patients are generally only used if the mechanism of action of a drug indicates that it will not be tolerated in healthy people – for example, if it induces pronounced hypoglycemia. They are primarily designed to assess the safety and tolerability of a drug, but the pharmacokinetics and, if possible, the pharmacodynamics are also measured.
The typical Phase I trial has a single ascending dose (SAD) design, meaning that subjects are dosed in small groups called cohorts. Each member of a cohort might receive a single dose of the study drug or a placebo. A very low dose is used for the first cohort. The dose is then escalated in the next cohort if safety and tolerability allow. Dose escalation is stopped when maximum tolerability and/or maximum exposure is reached.
SAD studies are usually followed by multiple ascending dose (MAD) studies, which have a very similar design, with cohorts and escalating doses. The only difference is that the subjects receive multiple doses of the study drug or placebo.
While safety and tolerability are still important endpoints, the multiple dose setting often allows first investigations of the pharmacodynamic effects in addition to the pharmacokinetics. Depending on the risk potential and the safety and tolerability revealed by the SAD study, many MAD studies may already involve patients rather than healthy individuals. That said, it is important to enroll a relatively healthy patient population with as few complications and concomitant diseases as possible.
Finally, food effect studies are often conducted to investigate the potential impact of food intake on the absorption of the drug. These studies are usually run as a crossover study, with volunteers being given two identical doses of the drug, one after fasting and one after a meal.Rate of Discovery of New Drugs Assignment Paper

Phase II

Phase II trials are performed on larger groups of patients and are designed to assess the efficacy of the drug and to continue the Phase I safety assessments. Most importantly, Phase II clinical studies help to establish therapeutic doses for the large-scale Phase III studies.
Phase II studies are sometimes divided into Phases IIA and IIB. Phase IIA is designed to assess dosing requirements whereas Phase IIB focuses on drug efficacy. The exact design of Phase II studies depends heavily on the compound’s mechanism of action. A proof-of-concept study should be included in Phase II if it has not already been done during the MAD-study in Phase I. In addition, a treatment study with several different doses of the compound in comparison with a placebo and/or an active comparator over a treatment duration of 12 to 16 weeks is usually an essential part of the Phase II program.

Preclinical development encompasses the activities that link drug discovery in the laboratory to initiation of human clinical trials. Preclinical studies can be designed to identify a lead candidate from several hits; develop the best procedure for new drug scale-up; select the best formulation; determine the route, frequency, and duration of exposure; and ultimately support the intended clinical trial design. The details of each preclinical development package can vary, but all have some common features. Rodent and nonrodent mammalian models are used to delineate the pharmacokinetic profile and general safety, as well as to identify toxicity patterns. One or more species may be used to determine the drug’s mean residence time in the body, which depends on inherent absorption, distribution, metabolism, and excretion properties. For drugs intended to treat Alzheimer’s disease or other brain-targeted diseases, the ability of a drug to cross the blood brain barrier may be a key issue. Toxicology and safety studies identify potential target organs for adverse effects and define the Therapeutic Index to set the initial starting doses in clinical trials. Pivotal preclinical safety studies generally require regulatory oversight as defined by US Food and Drug Administration (FDA) Good Laboratory Practices and international guidelines, including the International Conference on Harmonisation. Concurrent preclinical development activities include developing the Clinical Plan and preparing the new drug product, including the associated documentation to meet stringent FDA Good Manufacturing Practices regulatory guidelines. A wide range of commercial and government contract options are available for investigators seeking to advance their candidate(s). Government programs such as the Small Business Innovative Research and Small Business Technology Transfer grants and the National Institutes of Health Rapid Access to Interventional Development Pilot Program provide funding and services to assist applicants in preparing the preclinical programs and documentation for their drugs. Increasingly, private foundations are also funding preclinical work. Close interaction with the FDA, including a meeting to prepare for submission of an Investigational New Drug application, is critical to ensure that the preclinical development package properly supports the planned phase I clinical trial.Rate of Discovery of New Drugs Assignment Paper

In the United States, it takes an average of 12 years for an experimental drug to travel from the laboratory to your medicine cabinet. That is, if it makes it.

Only 5 in 5,000 drugs that enter preclinical testing progress to human testing. One of these 5 drugs that are tested in people is approved. The chance for a new drug to actually make it to market is thus only 1 in 5,000. Not very good odds.

The process of drug approval is controlled in most countries by a governmental regulatory agency. In the U.S., the Food and Drug Administration (FDA) governs this process. The FDA requires the following sequence of events before approving a drug.

Preclinical Testing: A pharmaceutical company conducts certain studies before the future drug is ever given to a human being. Laboratory and animal studies must be done to demonstrate the biological activity of the drug against the targeted disease. The drug must also be evaluated for safety. These tests take on the average 3 1/2 years.

 

Investigational New Drug Application (IND): The pharmaceutical company files an IND with the FDA to begin testing the drug in people. The IND becomes effective if the FDA does not disapprove it within 30 days. The IND must include the following information: the results of previous experiments; how, where and by whom the new studies will be conducted; the chemical structure of the compound; how it is thought to work in the body; any toxic effects found in the animal studies; and how the compound is manufactured. The IND must also be reviewed and approved by the Institutional Review Board where the studies will be conducted.

 

Phase I Clinical Trials: Phase I studies are usually the first tests of a drug under development in healthy volunteers. These studies involve about 20 to 80 volunteers. The tests determine a drug’s safety profile, including the safe dosage range, plus how the drug is absorbed, distributed, metabolized and excreted, and the duration of its action. Phase I trials take on the average 1 year.

 

Phase II Clinical Trials: These are slightly larger studies that are done in patients with the disease for which the drug is intended. This phase is usually designed to identify what are the minimum and maximum dosages. The trials generally involve 100 to 300 volunteer patients and are controlled in design. They are done to assess the drug’s effectiveness. Phase II typically takes about 2 years.Rate of Discovery of New Drugs Assignment Paper

Phase III Clinical Trials: These are the definitive, large randomized trials that are submitted to the FDA in order to obtain approval of a drug. This phase examines the effectiveness as well as the safety (adverse events) of the new drug. Phase III trials usually involve 1,000 to 3,000 patients in clinics and hospitals. Patients are usually asked a list of possible side effects, often derived from what was observed in phase II studies. Patients are also free to report any other side effects that occur while they are on the new drug or the placebo (the “sugar pill” that is given to a percentage of patients in a trial study). Phase III takes on the average 3 years.

 

New Drug Application (NDA): Following the Phase III Clinical Trials, the drug manufacturer analyzes all the data from the studies and files an NDA with the FDA (provided the data appear to demonstrate the safety and effectiveness of the drug). The NDA contains all of the data gathered to date about the drug. (An NDA typically consists of at least 100,000 pages.) The average NDA review time for new drugs approved in 1992 was close to 30 months (2 1/2 years).

 

Phase IV Studies: Phase IV is any organized collection of data from patients who are taking a drug that has already received approval from the FDA. In Phase IV studies, patients may check boxes on a list (as in phase III studies) or they may just report other symptoms. Phase IV studies are commonly called “post-marketing studies.”

As of this writing, there are more than 181,200 ongoing clinical studies throughout the United States and around the globe per the U.S. National Institutes of Health. However, since the Food and Drug Administration came into being in 1938 just 1,453 drugs have been approved through the end of 2013, according to the Regulatory Affairs Professionals Society. In other words, the success rate of experimental drugs making it to pharmacy shelves or your medicine cabinet is pretty low.

 

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Yet, if you think that’s low, you should see the success rate of drugs in preclinical studies making it into clinical studies. According to Medicine.net just five out every 5,000 preclinical drugs will see the light of day being tested on humans. Further, its statistics show that just one of those five will be approved by the FDA. If Medicine.net’s statistics hold water it would mean that nearly 7.3 million drugs have been reviewed in the preclinical setting just to net the aforementioned 1,453 FDA approved drugs since 1938. And people wonder why brand-name drugs are so pricey!Rate of Discovery of New Drugs Assignment Paper

How exactly is a drug developed?
The process is somewhat opaque, and I’d be willing to go on record saying that a vast majority of Americans and healthcare investors don’t quite understand the ins and outs of the process.

To that end we’ll clear things up here today and plainly discuss the nine step process most drugs will go through from concept to your medicine cabinet. I say most drugs since new processes have been recently developed, such as the breakthrough therapy designation, which can expedite the development and review of game-changing drugs. However, for the sake of simplicity I’ll leave that for another time and discussion.

Step 1: Drug discovery and target validation
The first step in the drug development process involves discovery work. This is where drug development companies choose a molecule, such as a gene or protein, to target with a drug. This is also where the drug developer will confirm that the molecule is indeed involved with the disease in question. After testing multiple drug molecules, the drug development company will choose those that have promise. Keep in mind it’s not uncommon for drug developers to have more than a handful of promising lead compounds at this stage.

Step 2: Preclinical testing
The next step in the drug development process is preclinical testing, which in itself is divided into two subcomponents: in vitro and in vivo testing. In vitro testing examines the drug molecules’ interactions in test tubes and within the lab setting. In vivo testing involves testing the drug molecules on animal models and in other living cell cultures. Although efficacy is beginning to be established here, safety is paramount as the FDA will not let preclinical studies move into human trials without extensive data on safety. This is the stage where researchers will whittle thousands of drug molecule candidates down to between one and five. By the time preclinical testing has concluded, many years have often passed.

Step 3: Investigational New Drug application filing
The third step involves submitting an Investigational New Drug application to the FDA prior to beginning human clinical trials. This is the point where the FDA will scrutinize the results from the preclinical testing, look at side effects and other safety features of an experimental drug, examine the drugs’ chemical structure and how it’s believed to work, and take its first look at the manufacturing process of the drug. If the FDA approves a drug developers’ IND then it can move onto human trials. An IND approval is also the point at which a patented drugs’ 20-year exclusivity period begins.Rate of Discovery of New Drugs Assignment Paper

Step 4: Phase 1 clinical studies
The first phase of human clinical testing involves a relatively small group of healthy people, usually a dozen to a few dozen, and it’ll focus entirely on safety. This stage of study involves looking at how a drug is absorbed and eliminated from the body, as well as what side effects it may cause and whether or not it’s producing the desired effect. Phase 1 clinical studies are also where maximum tolerated doses are established. It really is all about safety, although it’s not uncommon for drug developers to tout early signs of efficacy in phase 1. If everything looks promising the study moves to phase 2, or midstage trials.

Step 5: Phase 2 clinical studies
The two big changes between early stage and mid-stage trials are that the patient pool widens from a few dozen to perhaps 100 or more patients, and the patients being treated are no longer healthy volunteers but people being afflicted by the disease in question. Safety remains a big focus of phase 2 studies, with short-term side effects being closely monitored, although an increasing emphasis will begin to be placed on whether or not a drug is working as expected and if it’s improving the condition or not. Phase 2 studies also establish which dose (if multiple doses were tested, as is often the case) performed most optimally. If the experimental drug continues to look promising it’ll move onto late-stage studies.

Step 6: Phase 3 clinical studies
In phase 3 studies, safety remains a priority, but this is where efficacy also plays a big role. Phase 3 studies are designed by drug developers but approved by the FDA with guidelines for a clearly defined primary endpoint to determine the success or failure of a tested drug. Phase 3 trials involve even more patients, perhaps a few hundred to maybe thousands, and they are by far the longest and costliest of all components of the drug development process. This is also the stage where drug developers will begin to think about how they’re going to ramp up production if the phase 3 results are promising. Assuming an experimental drug meets its primary endpoint and is demonstrated to be safe, the next step is to file for its approval.

Step 7: New Drug Application filing
The seventh step in the drug development process is simple: filing a New Drug Application with the FDA. Unfortunately this isn’t just a single page that says “please look at our drug!” An NDA can be tens of thousands or perhaps 100,000 or more pages long, and it contains all research and safety data examined during each of the six prior steps. Still, this stage isn’t the point where the FDA has to make a decision to approve or deny the drug; it’s merely a stepping stone that says it promises to review the application over the next 10 months. If the NDA is accepted a PDUFA, or Prescription Drug User Fee Act, date is set 10 months down the road (for a standard application) whereby the FDA is expected to make its decision. Keep in mind the FDA can postpone this decision or even rule early should it choose.

Step 8: PDUFA date and decision
More often than not, the FDA will wait until the PDUFA date to release its decision. Essentially the FDA has three choices: it can approve a drug; it can outright deny a drug (which is pretty rare from what I’ve witnessed in 15 years), or it can request additional information by sending a complete response letter, or CRL. A CRL simply states what was lacking that prevented the drug from being approved and offers suggestions as to how to remedy the situation. Often times it requires drug developers to run additional studies or perhaps alter their manufacturing process to appease the FDA. If approved by the FDA, the drug becomes immediately available for commercial production.

Step 9: Phase 4 clinical studies
“Technically” an approved drug can make it to your medicine cabinet after step eight, but that doesn’t mean the drug developer is off the hook yet. Even after approval, it’s not uncommon for the FDA to request long-term safety studies be undertaken whereby drug developers are required to submit regular reports detailing any adverse events with the drug to the FDA. Even following approval, safety remains the top priority of the FDA.Rate of Discovery of New Drugs Assignment Paper

From start to finish, the entire drug development process (steps 1 through 8) usually spans about 10 to 15 years, leaving drug developers with around a decade or less of patent exclusivity on branded drugs once they make it to market. This should help provide some insight into why prescription drug prices are so high, why drug companies may seem like they’re taking “forever” in developing the next cure for a terrible disease, and why so few drugs actually earn a spot in your medicine cabinet.

Stages of Drug Development

Any drug development process must proceed through several stages in order to produce a product that is safe, efficacious, and has passed all regulatory requirements.

Pacific BioLabs can assist you through all stages of drug developoment. Our scientists can help you to determine your testing needs, and our experienced staff can perform the critical tests and studies that are necessary to win FDA approval.

To get you started, below we have provided an in-depth overview of many stages in the drug development process and necessary studies. Keep in mind this is just a guide; if you have any specific questions call Pacific BioLabs at 510-964-9000 to speak to a knowledgeable resource who can help you identify what testing you may need to perform.

Detailed Stages of Drug Development
Discovery
Product Characterization
Formulation, Delivery, Packaging Development
Pharmacokinetics And Drug Disposition
Preclinical Toxicology Testing And IND Application
Bioanalytical Testing
Clinical Trials

Discovery often begins with target identification – choosing a biochemical mechanism involved in a disease condition. Drug candidates, discovered in academic and pharmaceutical/biotech research labs, are tested for their interaction with the drug target. Up to 5,000 to 10,000 molecules for each potential drug candidate are subjected to a rigorous screening process which can include functional genomics and/or proteomics as well as other screening methods. Once scientists confirm interaction with the drug target, they typically validate that target by checking for activity versus the disease condition for which the drug is being developed. After careful review, one or more lead compounds are chosen.Rate of Discovery of New Drugs Assignment Paper

Product Characterization

When the candidate molecule shows promise as a therapeutic, it must be characterized—the molecule’s size, shape, strengths and weaknesses, preferred conditions for maintaining function, toxicity, bioactivity, and bioavailability must be determined. Characterization studies will undergo analytical method development and validation. Early stage pharmacology studies help to characterize the underlying mechanism of action of the compound.

Formulation, Delivery, Packaging Development

Drug developers must devise a formulation that ensures the proper drug delivery parameters. It is critical to begin looking ahead to clinical trials at this phase of the drug development process. Drug formulation and delivery may be refined continuously until, and even after, the drug’s final approval. Scientists determine the drug’s stability—in the formulation itself, and for all the parameters involved with storage and shipment, such as heat, light, and time. The formulation must remain potent and sterile; and it must also remain safe (nontoxic). It may also be necessary to perform leachables and extractables studies on containers or packaging.

Pharmacokinetics And Drug Disposition

Pharmacokinetic (PK) and ADME (Absorption/Distribution/Metabolism/Excretion) studies provide useful feedback for formulation scientists. PK studies yield parameters such as AUC (area under the curve), Cmax (maximum concentration of the drug in blood), and Tmax (time at which Cmax is reached). Later on, this data from animal PK studies is compared to data from early stage clinical trials to check the predictive power of animal models.Rate of Discovery of New Drugs Assignment Paper

Preclinical Toxicology Testing and IND Application

Preclinical testing analyzes the bioactivity, safety, and efficacy of the formulated drug product. This testing is critical to a drug’s eventual success and, as such, is scrutinized by many regulatory entities. During the preclinical stage of the development process, plans for clinical trials and an Investigative New Drug (IND) application are prepared. Studies taking place during the preclinical stage should be designed to support the clinical studies that will follow.

To that end, the Laboratory is working on ATHENA (or the Advanced Tissue-engineered Human External Network Analyzer) to help advance progress in this area. The purpose of the project is to create surrogate organs (currently the liver, heart, lung, and kidney) to provide realistic, cost-effective testing of various chemicals. It uses live human cells in an environment designed to mimic the human body as closely as possible.

As part of the research, a tissue-based artificial lung, called PuLmo (for Pulmonary Lung Model), even breathes so it can be used to test drugs, toxins, particles, and other agents. This latter work won an R&D 100 award. These awards are known as the “Oscars of Innovation.”

New medicines are constantly under development. However, the Food and Drug Administration (FDA) has strict safety protocols on the steps new drugs must go through before people can use them. Given that approximately 90 percent of pharmaceuticals fail their clinical trials despite passing animal studies, and there are thousands of chemicals whose effects on the human body are unknown, the ability to easily distinguish the harmful from the helpful would be extremely beneficial.Rate of Discovery of New Drugs Assignment Paper

To that end, the Laboratory is working on ATHENA (or the Advanced Tissue-engineered Human External Network Analyzer) to help advance progress in this area. The purpose of the project is to create surrogate organs (currently the liver, heart, lung, and kidney) to provide realistic, cost-effective testing of various chemicals. It uses live human cells in an environment designed to mimic the human body as closely as possible.

As part of the research, a tissue-based artificial lung, called PuLmo (for Pulmonary Lung Model), even breathes so it can be used to test drugs, toxins, particles, and other agents. This latter work won an R&D 100 award. These awards are known as the “Oscars of Innovation.”

Have you heard the oft-repeated “fact” that it takes at least 10 years from initial discovery for a new drug to enter the marketplace? Take it with a grain of salt. The drug development journey is closer to 30 years.

I’ve experienced the lag time between discovery and commercial success as the co-founder of a biotech startup, and now I study it at the Center for Integration of Science and Industry at Bentley University.

In the early 1990s, I co-founded GeneMedicine Inc., one of the first gene therapy companies. It had a successful startup, raised hundreds of millions of dollars, pioneered new product and business opportunities, and completed an initial public offering, which gave our initial investors a substantial profit.

This article provides a brief overview of the processes of drug discovery and development. Our aim is to help scientists whose research may be relevant to drug discovery and/or development to frame their research report in a way that appropriately places their findings within the drug discovery and development process and thereby support effective translation of preclinical research to humans. One overall theme of our article is that the process is sufficiently long, complex, and expensive so that many biological targets must be considered for every new medicine eventually approved for clinical use and new research tools may be needed to investigate each new target. Studies that contribute to solving any of the many scientific and operational issues involved in the development process can improve the efficiency of the process. An awareness of these issues allows the early implementation of measures to increase the opportunity for success. As editors of the journal, we encourage submission of research reports that provide data relevant to the issues presented.Rate of Discovery of New Drugs Assignment Paper

1. The process: Many years, many failures, much uncertainty

Most often, the development of a new medicine starts when basic scientists learn of a biological target (e.g., a receptor, enzyme, protein, gene, etc.) that is involved in a biological process thought to be dysfunctional in patients with a disease such as Alzheimer’s disease (AD). Here, we are considering the discovery and development of entirely new medicines, those with a mode of action different from already approved medicines and intended for a clinical indication that is not addressed by approved medicines. Better medicines that are iterative improvements on current medications are valuable as they may offer benefits over existing medications in terms of potency, safety, tolerability, or convenience, but they usually do not involve the manipulation of biological targets different from those directly affected by existing medications.

Analyses across all therapeutic areas indicate that the development of a new medicine, from target identification through approval for marketing, takes over 12 years and often much longer [1]. The cost to develop a New Molecular Entity (NME; a small molecule compound) or New Biological Entity (NBE; an antibody, protein, gene therapy, or other biological medicine) is certainly over $1 billion and, on average, has been estimated to be about $2.6 billion [2]. Fig. 1, adapted from Paul et al. [3], shows a schematic of the stages involved in developing a new medicine along with average times required for each stage and the approximate cost (in 2010 dollars) for each phase of development. Importantly, Fig. 1 also depicts the number of molecules that must be entered into each stage of development to, eventually, produce one new approved medicine. This figure is based on analyses across several therapeutic areas and includes data from development programs that are new iterations of existing medicines as well as those seeking medicines based on completely new targets or that aim for completely unprecedented therapeutic indications. It seems highly likely that the numbers in Fig. 1 greatly underestimate the numbers of molecules needed at each stage of development to produce a new medicine to treat a disease for which no therapy currently exists. Separate figures for AD drug development programs are not available, but the last line of Fig. 1 shows clinical transition probabilities calculated by Cummings et al. [4], who reviewed all of the 244 unique compounds studied in clinical trials for AD from 2002 through 2012. It is evident that the likelihood of advancing an AD drug candidate has been very low when compared with those for development programs across a broad range of therapeutic areas. Stated another way, the probability is very low that any new biological target or molecule identified as potentially relevant to the modification of AD will result in an approved new medicine. We should anticipate that a very large number of biological targets will need to be discovered and interrogated pharmacologically and genetically to achieve a single new disease-modifying medicine for AD.Rate of Discovery of New Drugs Assignment Paper

In accordance with this, central nervous system (CNS) drugs have lower success rates and take a longer time to develop, than do other drug classes. Specifically, the success rate of neuropsychiatric drug candidates who enter into human testing to effectively reach the marketplace is dramatically lower (8.2%) than for all drugs combined (15%) [5], [6]. In the case of drugs focused toward AD progression, of the numerous evaluated clinically, the attrition rate has thus far been 100%. Furthermore, the average clinical development time for neuropsychiatric drugs is in the order of 8.7 years, as compared with 5.9 years for antiviralagents, almost 50% longer. The time required to gain regulatory approval is also longer for neurological drugs, 1.9 years as opposed to an average of 1.2 years for all drugs. Taking into account the approximately 6 to 10 years that drugs generally are in the preclinical phase of development, neurological drugs can take up to 18 years to run the gauntlet from initial laboratory evaluation to regulatory approval and use [5], [6]—a long duration in relation to the current 20-year patent protection rights. The drug development process is set up, particularly at the stage of clinical development, to “fail fast, fail early” in a strategy to eliminate key risks before making a expensive late-stage investment [7], [8]. Nevertheless, neurological agents tend to fail later during the clinical development process—in phase 3 clinical trials [5], [6], particularly for recent AD experimental therapeutics, thereby making CNS drugs among the most expensive to develop. It is hence important to optimize each piece of the preclinical and clinical development process.

2. Discovery: From target to clinical candidate

The goal of a preclinical drug discovery program is to deliver one or more clinical candidate molecules, each of which has sufficient evidence of biologic activity at a target relevant to a disease as well as sufficient safety and drug-like properties so that it can be entered into human testing. Most discovery programs seek to produce more than one candidate molecule because, as is shown in Fig. 1, many molecules do not move through the entire process because of problems with safety, kinetics, potency, intellectual property protection, or other factors. There is no simple formula for producing a viable clinical candidate molecule, although extensive collaboration of chemistry, biology, toxicology, and pharmacokinetics is almost universally the norm in modern drug discovery programs [9]; small molecule drug discovery programs typically produce massive amounts of data using high-throughput screening techniques that evaluate many compounds at many doses against many assays [9].Rate of Discovery of New Drugs Assignment Paper

Some of the information that should be developed during discovery studies for a clinical candidate molecule is shown in Fig. 2. All of the topics listed in this figure will need to be addressed before deciding whether a molecule is suitable for testing in humans. There are no perfect discovery programs, and some of the desired information listed in Fig. 2 may be missing; however, gaps in knowledge at this stage often lead to difficulties in interpreting later studies. Critical to moving any molecule forward will be an assessment of target validity; that is, does the molecule target an aspect of biology that is relevant to the disease of interest? And, is the target expressed in the human brain during the disease process that allows a window of opportunity for treatment? Target validation has no uniformly accepted definition, although data from humans showing some relationship of the proposed target to the disease, such as AD, are essential. For potential medicines that are designed to be improved iterations on already approved medicines, the validating data are usually quite compelling and derive from the fact that other medicines with similar mechanism of action have shown efficacy. For AD, where there is no disease-modifying medicine, such validation is not available. In the search for medicines directed toward completely novel targets, advances in genetics, such as the Human Genome Project, have produced many potential new pharmacological targetsand genetic “validation” is often cited as a reason for pursuing a novel drug target [10]. However, mechanistic targets such as receptors and enzymes that are well understood biologically have led to many of the medicines currently used; in addition, whole animal models that reproduce some physiological aspects of human disease such as abnormal activity in a specific neural circuit have also been used successfully [10]. None of these approaches to target validation are a guarantee of success in screening for potential new medicines, but it is important to be very explicit about the data supporting the pursuit of a target and the kinds of screening tools available for identifying potential clinical candidates. This explicit understanding will help insure that results obtained with one molecule can be used to help inform the development program for the next molecule.

This article provides a brief overview of the processes of drug discovery and development. Our aim is to help scientists whose research may be relevant to drug discovery and/or development to frame their research report in a way that appropriately places their findings within the drug discovery and development process and thereby support effective translation of preclinical research to humans. One overall theme of our article is that the process is sufficiently long, complex, and expensive so that many biological targets must be considered for every new medicine eventually approved for clinical use and new research tools may be needed to investigate each new target. Studies that contribute to solving any of the many scientific and operational issues involved in the development process can improve the efficiency of the process. An awareness of these issues allows the early implementation of measures to increase the opportunity for success. As editors of the journal, we encourage submission of research reports that provide data relevant to the issues presented.Rate of Discovery of New Drugs Assignment Paper

1. The process: Many years, many failures, much uncertainty

Most often, the development of a new medicine starts when basic scientists learn of a biological target (e.g., a receptor, enzyme, protein, gene, etc.) that is involved in a biological process thought to be dysfunctional in patients with a disease such as Alzheimer’s disease (AD). Here, we are considering the discovery and development of entirely new medicines, those with a mode of action different from already approved medicines and intended for a clinical indication that is not addressed by approved medicines. Better medicines that are iterative improvements on current medications are valuable as they may offer benefits over existing medications in terms of potency, safety, tolerability, or convenience, but they usually do not involve the manipulation of biological targets different from those directly affected by existing medications.

Analyses across all therapeutic areas indicate that the development of a new medicine, from target identification through approval for marketing, takes over 12 years and often much longer [1]. The cost to develop a New Molecular Entity (NME; a small molecule compound) or New Biological Entity (NBE; an antibody, protein, gene therapy, or other biological medicine) is certainly over $1 billion and, on average, has been estimated to be about $2.6 billion [2]. Fig. 1, adapted from Paul et al. [3], shows a schematic of the stages involved in developing a new medicine along with average times required for each stage and the approximate cost (in 2010 dollars) for each phase of development. Importantly, Fig. 1 also depicts the number of molecules that must be entered into each stage of development to, eventually, produce one new approved medicine. This figure is based on analyses across several therapeutic areas and includes data from development programs that are new iterations of existing medicines as well as those seeking medicines based on completely new targets or that aim for completely unprecedented therapeutic indications. It seems highly likely that the numbers in Fig. 1 greatly underestimate the numbers of molecules needed at each stage of development to produce a new medicine to treat a disease for which no therapy currently exists. Separate figures for AD drug development programs are not available, but the last line of Fig. 1 shows clinical transition probabilities calculated by Cummings et al. [4], who reviewed all of the 244 unique compounds studied in clinical trials for AD from 2002 through 2012. It is evident that the likelihood of advancing an AD drug candidate has been very low when compared with those for development programs across a broad range of therapeutic areas. Stated another way, the probability is very low that any new biological target or molecule identified as potentially relevant to the modification of AD will result in an approved new medicine. We should anticipate that a very large number of biological targets will need to be discovered and interrogated pharmacologically and genetically to achieve a single new disease-modifying medicine for AD.Rate of Discovery of New Drugs Assignment Paper

In accordance with this, central nervous system (CNS) drugs have lower success rates and take a longer time to develop, than do other drug classes. Specifically, the success rate of neuropsychiatric drug candidates who enter into human testing to effectively reach the marketplace is dramatically lower (8.2%) than for all drugs combined (15%) [5], [6]. In the case of drugs focused toward AD progression, of the numerous evaluated clinically, the attrition rate has thus far been 100%. Furthermore, the average clinical development time for neuropsychiatric drugs is in the order of 8.7 years, as compared with 5.9 years for antiviralagents, almost 50% longer. The time required to gain regulatory approval is also longer for neurological drugs, 1.9 years as opposed to an average of 1.2 years for all drugs. Taking into account the approximately 6 to 10 years that drugs generally are in the preclinical phase of development, neurological drugs can take up to 18 years to run the gauntlet from initial laboratory evaluation to regulatory approval and use [5], [6]—a long duration in relation to the current 20-year patent protection rights. The drug development process is set up, particularly at the stage of clinical development, to “fail fast, fail early” in a strategy to eliminate key risks before making a expensive late-stage investment [7], [8]. Nevertheless, neurological agents tend to fail later during the clinical development process—in phase 3 clinical trials [5], [6], particularly for recent AD experimental therapeutics, thereby making CNS drugs among the most expensive to develop. It is hence important to optimize each piece of the preclinical and clinical development process.

2. Discovery: From target to clinical candidate

The goal of a preclinical drug discovery program is to deliver one or more clinical candidate molecules, each of which has sufficient evidence of biologic activity at a target relevant to a disease as well as sufficient safety and drug-like properties so that it can be entered into human testing. Most discovery programs seek to produce more than one candidate molecule because, as is shown in Fig. 1, many molecules do not move through the entire process because of problems with safety, kinetics, potency, intellectual property protection, or other factors. There is no simple formula for producing a viable clinical candidate molecule, although extensive collaboration of chemistry, biology, toxicology, and pharmacokinetics is almost universally the norm in modern drug discovery programs [9]; small molecule drug discovery programs typically produce massive amounts of data using high-throughput screening techniques that evaluate many compounds at many doses against many assays [9].

Some of the information that should be developed during discovery studies for a clinical candidate molecule is shown in Fig. 2. All of the topics listed in this figure will need to be addressed before deciding whether a molecule is suitable for testing in humans. There are no perfect discovery programs, and some of the desired information listed in Fig. 2 may be missing; however, gaps in knowledge at this stage often lead to difficulties in interpreting later studies. Critical to moving any molecule forward will be an assessment of target validity; that is, does the molecule target an aspect of biology that is relevant to the disease of interest? And, is the target expressed in the human brain during the disease process that allows a window of opportunity for treatment? Target validation has no uniformly accepted definition, although data from humans showing some relationship of the proposed target to the disease, such as AD, are essential. For potential medicines that are designed to be improved iterations on already approved medicines, the validating data are usually quite compelling and derive from the fact that other medicines with similar mechanism of action have shown efficacy. For AD, where there is no disease-modifying medicine, such validation is not available. In the search for medicines directed toward completely novel targets, advances in genetics, such as the Human Genome Project, have produced many potential new pharmacological targetsand genetic “validation” is often cited as a reason for pursuing a novel drug target [10]. However, mechanistic targets such as receptors and enzymes that are well understood biologically have led to many of the medicines currently used; in addition, whole animal models that reproduce some physiological aspects of human disease such as abnormal activity in a specific neural circuit have also been used successfully [10]. None of these approaches to target validation are a guarantee of success in screening for potential new medicines, but it is important to be very explicit about the data supporting the pursuit of a target and the kinds of screening tools available for identifying potential clinical candidates. This explicit understanding will help insure that results obtained with one molecule can be used to help inform the development program for the next molecule.Rate of Discovery of New Drugs Assignment Paper

When we look back at our past records about discovering new drugs, it is dismal and pathetic. Leave aside the past, the future does not seem to be rosy considering the present conditions. As a growing world economy, are we doing enough in this direction? Future’s seeds are always sown in the past. Have we done enough or are we still doing enough to be optimistic of a new era where India would be a fountain head of innovations? There are so many questions which need to be answered. A critical analysis of the past and the present becomes pertinent to get answers of these questions.

New drug discovery is a complex process involving the inputs of many branches of science like, physical and organic chemistry, cellular and molecular biology, biochemistry, pharmacology, computational chemistry, and so on and so forth. Discovering a new drug molecule and bringing it to the clinician’s desk requires team efforts of so many experts in their own field. When we peep into the past we notice that new drugs have been discovered either serendipitously or by keen observations and systematic studies. Upto 1980s the rate of launching new drug molecules per year has remained impressive but 1990 onwards this rate has decreased dramatically. Medicinal chemists became more rational in their approach in the later years for discovering new drugs and that should have increased the rate of discovering new drug molecules over the years. But astonishinglythe reverse has happened. One understandable reason is a stricter regulatory regime which is more sensitive to the reporting of side effects of the new drugs and providing of exhaustive pre-clinical and clinical data. The other reason scientists quote is the lack of information about new disease targets. Hype was created about human genome mapping that once completed this would provide immense amount of information about new biological targets for discovering new drug molecules. Alas!this has not happened. There is no denying the fact that fewer drugs are getting discovered across the globe, but what is the situation in our country?

Free India took its first breath in the lap of utter poverty. Even food and other essential commodities were not available to its people leave aside medicines. To provide essential drugs at cheaper rates to the people, Government of India enacted a legislation, the Indian Patent Act, in 1970. According to this Act “Product Patenting” was not tenable as this Act recognized only the “Process Patenting.” This Act proved to be a boon for the growth of pharmaceutical industry in the country. By modifying the processes for the active pharmaceutical ingredient APIs as well as the formulations, medicines could be offered to the poor populace at affordable prices. Pharmaceutical industry grew by leaps and bounds and India became an exporting country of pharmaceuticals even to the Western countries.Rate of Discovery of New Drugs Assignment Paper

Unfortunately, this legislation which catalyzed the growth of pharmaceutical industry and provided medicines at affordable rates to the poor people, proved to be a big stumbling block in the new drug discovery process for the nascent pharmaceutical industry. It crippled the basic research in pharmaceutical field and forced us to become copycats. By adopting reverse engineering we could make any molecule in the world by modifying the process. This proved to be a death knell for our basic research. When a new drug molecule was available at the least price by slight modification of the available synthetic process then why would anybody spend fortunes in discovering new drug molecules? Apart from this main reason, lack of team work is another factor why we are not succeeding even now in our efforts. But, is discovery of new drug molecules such a cumbersome resource and time consuming process that it seems to be a dream for us? That too, when we are ushering into an era, which the whole world says, belongs to India and China. To my mind, discovering a new drug entity is not as difficult and unachievable a task as it is being projected. May be we are being misled by certain forces with ulterior motives and selfish interests so that we do not make serious efforts in this direction.

As mentioned earlier new drug discovery requires team work. We do not know when we will start working as a team. Another misconception people have is about computer aided drug designing or molecular modeling. People think that drug designing techniques will provide new drug molecules without efforts. This has not happened till date even after implementation of these techniques for the last 10-15 years. And this will not happen in near future also. Computer aided drug designing techniques are lead optimization techniques and in some cases hasten the process of lead identification. These techniques are used to make the drug discovery process more rational. On their own these will never discover a new drug. But yes, these techniques save a lot of time and energy and provide us a better understanding of the discovery process. There are many techniques which are part of computer aided drug designing process. To start with the simplest one is Quantitative structure–activity relationship (QSAR) which is also referred to as 2D-QSAR sometimes. 3D-QSAR involving Comparative Molecular Field Analysis (CoMFA) and Comparative molecular similarity index analysis (CoMSIA) are extension of QSAR. QSAR is not able to take the three dimensional structure of a molecule into consideration due to absence of three-dimensional parameterization of structures. 3D-QSAR scores over QSAR in this respect. Docking studies throw more light on the binding modes of drugs with their target proteins but it is feasible only when the crystal structure of the target enzyme/protein is known with good resolution. Docking studies are also used for virtual screening of databases. But the ideal technique for virtual screening of compounds is through pharmacophore mapping and screening, especially when the structure of the target is not known. Very large databases can be first screened by pharmacophorebecause the technique is quite fast followed by screening of the positive hits using docking studies. Insilico designing of novel compounds can also be performed using deNovo designing techniques subject to the condition that the target structure in known.

Drug discovery scenario has completely changed in the last decade. Earlier, a lot of emphasis used to be put in discovering more potent compounds with fewer side effects using animal models. When such compounds were entering the clinical phase, these used to face a high rejection rates due to absorption, distribution, metabolism, excretion and toxicity (ADMET) problems thereby putting huge efforts and resources down the drain. Nowadays more emphasis is on assessing new chemical entities for ADMET parameters prior to further studies. Several software are available to theoretical assessing of ADMET parameters for a new chemical entity. In a typical situation when a new chemical entity shows good binding affinity with the target enzyme/receptor, it is assessed for its ADMET parameters followed by whole body autoradiography. Further biological evaluations are performed only when the compound passes these litmus tests.Rate of Discovery of New Drugs Assignment Paper

It is a pity that majority of our researchers in the field are unaware about these techniques. We are not making serious efforts to train our graduate students in these techniques. There is a need to give enmass training to our researchers on computer aided designing techniques. In 1980s several groups were working in the field of QSAR in Pharmacy institutions in India. Those days the number of pharmacy education imparting institutions were also limited, may be 20-25 at the most. Today, there are about a thousand such institutions but the number of research groups engaged in new drug discovery can be counted on fingers. And how many of these research groups are using computer-aided designing tools? The figure has awfully deteriorated. Where are we heading to? Have we put ourselves in a reverse gear?

Stagnation in new drug research and development has been a focal issue in past decades. Although the number of US Food and Drug Administration (FDA) approvals of new molecular entities (NMEs) hit a 21‐year high in 2017, such approvals have remained at low levels since the 2000s, after reaching a peak in the mid‐1990s.1, 2Despite various initiatives and efforts to improve success rates in both the public and private sectors, the likelihood of approvals (LOAs) from phase I clinical trials remains at ~10%.3, 4 Challenging environmental conditions in research and development (R&D) have inevitably led the pharmaceutical industry to spend more on new drug development operations; R&D investment in 2014 tripled that of 1995, and doubled the amount spent in 2000.5

The decreasing numbers of NME approvals and increasing R&D costs have often been discussed in relation to the exhaustion of obvious drug targets. Hopkins and Groom6showed that there were 600–1500 “drug targets” that could potentially receive industrial research. The creation of innovative new drugs is predicated on the discovery and development of new technologies in addition to the search for new targets outside the existing sphere, which is often achieved by linking industrial drug discovery to basic academic research. Basic research in the context of pharmaceutical R&D, which typically entails target‐screening and in vitro studies based on novel concepts, is a strength of academic institutions.7 The process of drug discovery in academia also differs from that of the industry in that academic researchers tend to pursue higher‐risk targets and undertake more in‐depth research than industrial researchers.8

Kneller9 analyzed the origins of 252 drugs that received FDA approval between 1998 and 2007. Of the 252 compounds, 191 originated from pharmaceutical companies, whereas the remaining 61 originated from universities and biotechnology companies before being transferred to the industry.9 The analysis showed that universities and biotechnology companies substantially contributed to the current discovery of innovative drugs during the period of study, and discussed how drug “seeds” that originated from universities and biotechnology companies were transferred to the industry.

Given the concerns about possible stagnation regarding the successful drug discovery and development projects that originate from within the industry, pharmaceutical companies around the world have been pursuing open innovation in an attempt to acquire drug discovery “seeds” that originate in academia.10, 11 It is of research interest whether such strategic options have actually resulted in new drug development successes. There have been various studies on the historical performance of drug discovery and development in both academia and the public sector.7, 12, 13, 14 Most of these studies involved questionnaire surveys that were only conducted for the examination of approved drugs. This is because it is practically difficult to grasp the details of projects that failed or were dropped at some stage of the R&D process. Considering the important role of academia in finding and/or creating seeds through fundamental research in the early stages of development, we need to focus not only on the successful portions of drugs, but on all drug candidates and programs during a specific period.Rate of Discovery of New Drugs Assignment Paper

The objective of our study was to provide a broad overview of performance in addition to the characteristics of current academic drug discovery and development in the United States. In this study, academic drug discovery and development was defined as the drug discovery and development projects involving compounds that originate in academia. We conducted an analysis on projects conducted by major research universities in the United States and examined how academic‐industrial collaboration was associated with success rates in each clinical trial stage.

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Methods

We chose projects for which we could confirm the success or failure of nonclinical trials at 36 universities in the United States. The universities were selected according to their rankings in “The 2014–2015 Times Higher Education World University Rankings’ clinical, preclinical, and health.”15 The names of the 36 universities are provided in Table S1.

Information on candidate compounds was extracted from the Cortellis Competitive Intelligence (Clarivate Analytics) as of September 9 and September 10, 2015. We found 798 projects for which nonclinical research was begun between 1991 and 2010, and for which information on the success or suspension of nonclinical research was available by 2015. The beginning year of nonclinical research was estimated based on the methods used by Paul et al.16. We excluded compounds originally discovered by universities, but for which biotechnology companies were specified as the originator. We included all types and modalities of drugs in this study.

In view of the scarcity of new antimicrobial medications being found from terrestrial environment, there is an urge to explore different environmental sources. The marine environment consists of a wide diversity of microorganisms, and thus the discovery of many medically useful compounds. The antivirals acyclovir and cytarabine used for herpes virus and non-Hodgkin’s lymphoma respectively were originally isolated from marine sponges, showing the potential of marine life as a novel source of medicines (1). As reported in a study conducted in South East coast of India, organisms originated from the genera Vibrio, Pseudomonas, Marinobacter and Bacillus have been isolated and proven to exhibit antimicrobial activity, where the isolate belonged to Bacillus species had the highest activity against Bacillus subtilis, E. coli and C. albicans (13). Besides that, aquatic actinomycetes have also been reported to produce bioactive compounds with potential clinical uses such as salinosporamide-A (from Salinispora sp.), marinopyrroles (from Streptomyces sp.) and marinobactin (from Marinobacter sp.) (14-16). Antibacterial actinomycetes have been successfully isolated from Lake Tana, Ethiopia, of which 13 isolates showed antibacterial ability against at least one of the tested bacteria, such as K. pneumoniae, S. aureus, P. aeruginosa and E. coli (17). Similarly, a study conducted in Ghana has isolated 27 antibiotic-producing microorganisms from marine and freshwater sources and it has been found that one of the active isolates produced metabolites with a broad antibacterial activity against both tested Gram-positive and Gram-negative bacteria (18).

Although it is apparent that the ocean is a boundless source for novel antibacterial compounds, the water sources remain underexplored and unexploited. The aim of this study was to isolate antibiotic-producing microorganisms from the aquatic environment around Bath area and to determine their antibacterial activity against six test bacteria, three of which belonged to Gram-positive bacteria and the other three were Gram-negative bacteria.Rate of Discovery of New Drugs Assignment Paper

2. Methods and Materials

2.1 Sample collection

Water samples with sediments were collected from River Avon, Bath City Farm, Rainbow Wood Farm and pond in the University of Bath.

2.2 Isolation of microorganisms

The water samples were serially diluted up to 10-4 in phosphate buffered saline (PBS) (Oxoid). Each diluted sample was inoculated into media by spread plate technique and incubated at 28°C. Three different media were used, namely minimal salt agar (Sigma), nutrient agar (Oxoid) and tryptic soy agar (TSA) (Oxoid).

2.3 Screening of isolates for antibacterial activity

Crowded plate technique was used to screen microorganisms with antagonistic activity. Inhibition activity can be demonstrated by the formation of clear zones surrounding the colonies, after being incubated for three days. Colonies of interest were selected and repeated streaking technique was used to purify the isolated colonies.

An overlay of soft agar with a concentration of 0.75% was performed using double-layer agar technique to confirm the antibacterial activity of the isolates. 10ml of soft agar maintained in a water bath at 42°C was mixed with 100l of methicillin-sensitive Staphylococcus aureus (MSSA) Newman (19) before pouring it onto the solid agar. Clear zones produced indicated the synthesis of compounds active against the test bacterium.

Perpendicular streak test was carried out where isolates were streaked as a single line along the diameter of TSA and incubated at 37°C and 28°C for one day each or 28°C for three days. The test organisms were then cross streaked at right angles to the original streak of isolates. The test organisms used included MSSA Newman, methicillin-resistant Staphylococcus aureus (MRSA) 252 (20), Enterococcus faecium (E. faecium) E1162 (21), Escherichia coli (E.coli) BW25113 (22), Pseudomonas aeruginosa (P. aeruginosa) PA01 (23), and Klebsiella pneumoniae (K. pneumoniae) (departmental strain collection). The plates were incubated at 37°C for 24 hours and the length of zones of inhibition was measured. Control plates without isolates of interest were simultaneously streaked with test organisms to study their normal growth.

For isolates that showed activity against E. coli BW25113, they were further tested with four other E. coli strains: E. coli ER2420 (pACYC184) (24), E. coli ER2420 (pACYC177) (24), E. coli SURE (pET-Amy) (25), and E. coli XL1Blue (pSG1164) (26), which displayed different antibiotics resistance. Perpendicular streak test was carried out to identify the antibiotic(s) produced.

2.4 Antibacterial activity of supernatant

Five isolates were selected and grown in the nutrient broth (NB) (Oxoid) for three days. The supernatant was obtained by centrifugation at 4 °C, 5000 g for 20 minutes and it was filter sterilised before use. The antibacterial activity of the supernatant of each isolate was investigated as described:

(i) 100l of the supernatant of each isolate was mixed with 100l of each test organism culture (106 cfu/ml) and plated in 96-well plates. Eight replicates were done for each isolate. 200l of NB served as a negative control, while a mixture of 100l of each test organism culture and 100l of NB served as a positive control. The plates were incubated at 37°C for 24 hours and the OD of the mixture was measured.

(ii) Agar well diffusion test was carried out where 100l of each test organism culture (106 cfu/ml) were plated on TSA and left for 30 minutes. Five wells of 9mm were punched in the TSA with a sterile cork borer and 100l of each supernatant sample was filled into the wells of each plate inoculated with different test organisms. The plates were incubated at 37°C for 24 hours and observed for growth inhibition zones.Rate of Discovery of New Drugs Assignment Paper

2.5 Characterisation of colonies of interest

The strains of interest were characterised using gram staining to identify their classes, shapes and sizes under a light microscope.

2.6 Genomic DNA extraction

Genomic DNA of each isolate was extracted by alkaline lysis as described in (27; 28) with some modifications. In this experiment, a loopful of cells was emulsified in 20l lysis buffer (0.25% sodium dodecyl sulfate (SDS) (SIGMA), 0.05N NaOH (Fisherbrand)) and heated at 95°C for 15 minutes (28). The cells were then centrifuged at 13,000rpm for 30 seconds and the supernatant was diluted with 180l sterile water and stored at -20°C for future use.

2.7 PCR primers and conditions

Five isolates of interest were chosen for 16S rRNA sequencing. The 16S rRNA gene was amplified by PCR using forward primer 27F (5′- AGAGTTTGATCMTGGCTCAG-3′) (Sigma) and reverse primer 1492R (5′-TACGGYTACCTTGTTACGACTT-3′) (Sigma). PCR was conducted with One Taq @ Standard Reaction Buffer (5x) (New England Biolabs Inc.) using manufacturer’s instruction.

PCR protocol was as follow: at 94°C for 2 min followed by 30 cycles at 94°C for 30 s, 55°C for 60 s, and 68°C for 90 s, then 68°C for 5 min in a single step. The samples were then held at 4°C. The PCR products were washed using Monarch PCR & DNA Cleanup Kit (5g) (New England BioLabs Inc.) before performing agarose gel electrophoresis to evaluate their purity and quality.

2.8 Agarose gel electrophoresis

Visualisation of the PCR products was carried out on 1% agarose-TAE gel (Invitrogen, ThermoScientific) as described in (27) and Bioline Hyperladder I 100lanes was used as the ladder. Following the result of electrophoresis, Mix2Seq protocol was followed to prepare the samples for 16S rRNA sequencing.

2.9 Statistical analysis

Student t-test was used to calculate the significance of the difference between the experimental and the control samples. Differences were considered significant at p<0.05. 3. Results 3.1 Isolation and screening of microbial isolates A total of 106 colonies were found to show inhibition of the growth of the surrounding microorganisms. The majority of the active isolates were collected from Bath City Farm while the least was collected from River Avon. It was also shown that most of the isolates of interest grew better on TSA compared to NA. Following primary isolation, the isolates were overlaid with MSSA to confirm their antibacterial activity as shown in Figure 1. Out of the 106 colonies, only 34 of them inhibited the growth of MSSA and they were further investigated through perpendicular streak test with six bacteria. The zone of inhibition from perpendicular streaking of the isolates indicated antibiotic-producing activity as depicted in Figure 2. From the 34 isolates, only 11 isolates showed good outcomes, five independent repeats were done and the results are shown in Table 1. The average length of the inhibition zone produced by the isolates varied greatly, depending on the susceptibility of the test organisms to the antibiotics produced. In the perpendicular streak test, MSSA, which served as a control, was inhibited by all of the tested isolates. It was found that none of the isolates was active against P. aeruginosa, while MRSA was sensitive to every antibiotic-producing isolate. The number of isolates that inhibited the growth of E. coli and E. faecium was six and five respectively, and only  Rate of Discovery of New Drugs Assignment Paper Figure 1. An overlay of MSSA was performed on TSA to determine the antibacterial activity of the isolates. A prominent zone of inhibition was shown by isolate 44, where the growth of MSSA was inhibited. three of the isolates antagonised the growth of K. pneumoniae. Out of the eleven isolates, five isolates were active against all three Gram-positive test organisms, three of which were active only against Gram-positive bacteria. Nine isolates demonstrated inhibition on at least three of the test organisms, of which five isolates inhibited four test organisms in total, and only two isolates inhibited two test organisms. The isolate 107 showed the most potent antagonistic activity against the test organisms as the lengths of inhibition observed were the longest among all (20.8  6.4 mm, 24.3  5.6 mm, 16.2  7.6 mm and 13.6  4.7 mm against MSSA, MRSA, E. faecium and E. coli respectively), while the isolate 103 exhibited the least potent Figure 2. Perpendicular streak method was used to study the activity of the isolates against six bacteria. Colony 104 demonstrated clear inhibition of the growth of MSSA, MRSA and E. faecium. antagonistic ability with the weakest inhibition against the test organisms MSSA, MRSA and E. coli (4.4  2.2 mm, 4.0  1.2 mm and 6.0  1.4 mm). MSSA and MRSA were both the Gram-positive bacteria most commonly inhibited by the isolates while E. coli was the most commonly inhibited Gram-negative bacterium among all. 3.2 Antibacterial activity of the supernatant Two tests were carried out to study the antibacterial activity of the supernatant of each isolate: (i) In the agar well diffusion test, the diameter of the zone of inhibition was measured and recorded. A fairly weak zone of inhibition was shown by all isolates, ranging from 9.5mm to 12.5mm. Isolate 18 was active against all bacteria, also demonstrating the strongest inhibition among all, with a maximum zone of inhibition of 12.5  0.7 mm shown against E. faecium. Isolate 71 had a fairly low activity against the test organisms E. coli (10.2  0.7mm), P. aeruginosa (10  0mm) and K. pneumoniae (11.5  2.1mm), and showed no effect against any of the Gram-positive bacteria. The supernatant of isolates 102, 104 and 107 exhibited some antibacterial activity against both Gram-positive and Gram-negative bacteria, although the activity was weak (<2mm zone of inhibition). (ii) In the 96-well assays, the OD of the isolates in test organisms' broth was compared to the OD of the positive controls as shown in Figure 3. The reduction in OD indicated that there was antibacterial activity, whereas a similar reading of OD showed that there was no inhibition. It was shown that isolates 18, 71 and 102 demonstrated slight antagonistic activity against MSSA (only 102 showed significance with p=0.038), isolate 104 did not seem to have a great effect on MSSA while 107 had a mild promoting effect on the growth of MSSA (p=0.111).Rate of Discovery of New Drugs Assignment Paper All tested isolates antagonised MRSA and E. coli. For MRSA, the differences in the OD were significant, showing a significant inhibition effect from each isolate. The strongest inhibition against MRSA was shown by isolate 71 with a difference of 0.207 (90%) in the OD while isolate 18 showed the strongest activity against E. coli, giving a difference of 0.441 (97%) in the OD. Isolate 107 was less active against both MRSA and E. coli, giving the minimum zone of inhibition in both tests. There was no big difference in the OD shown among the isolates tested with E. faecium, except that isolate 18 was shown to have promoted the growth of the test organism, with a difference of 0.031 in the OD but it was not significant (p= 0.122). Isolates 18, 104 and 107 demonstrated slight enhancing effect while the other two isolates had an inhibitory effect on P. aeruginosa. All p values except for 104 are <0.05. A prominent inhibitory effect has been shown by isolate 18 against K. pneumoniae (p=0.0009), and isolate 71 gave a reduction in the OD (p=0.086). Three other isolates demonstrated a slight promoting effect on the growth of K. pneumoniae (p values >0.05).

As a whole, the supernatant of isolate 71 displayed the strongest activity against all test bacteria, while isolate 18 gave the greatest degree of zone inhibition, especially against MSSA, E. coli and K. pneumoniae.

3.3 Determination of the types of antibiotics produced by the isolates of interest

Three isolates that displayed antagonistic effect against E. coli were further screened against four different strains of E. coli to determine the possible antibiotic(s) produced. Isolates 18 showed powerful inhibition against all strains of E. coli, with every zone of inhibition exceeding 10mm. Isolate 107 inhibited the growth of every strain tested, with a zone of inhibition ranging from 5mm to 15mm. The

strongest inhibition was shown against E. coli SURE (pET-Amy) while the weakest inhibition was shown against E. coli BW 25113. This indicated that the antibiotics produced were not any of the antibiotics that the tested E. coli was resistant to. On the other hand, isolate 71 inhibited the growth of four E. coli strains, except for E. coli ER2420 (pACYC184), showing that one of the antibiotics produced may be kanamycin.

Table 2. E. coli strains and their respective antibiotic resistance

Strains of E. coli Antibiotic resistance

E. coli BW25113 None

E. coli ER2420 (pACYC184) Chloramphenicol, tetracycline

E. coli ER2420 (pACYC177) Ampicillin, kanamycin

E. coli XL1Blue (pSG1164) Ampicillin, tetracycline, chloramphenicol

E. coli SURE

(pET-Amy) Ampicillin, kanamycin, tetracycline

3.4 Gram stain

11 active isolates with promising antibacterial activity were gram stained. It was found that five isolates of interest belonged to Gram-negative bacteria, whereas the other six isolates belonged to Gram-positive bacteria. Among the five Gram-negative isolates, four of them were short rods, while isolate 36 had the shape of coccobacilli. On the other hand, all Gram-positive isolates were rod-shaped and half of the Gram-positive isolates were thin rods.

3.5 Visualisation of PCR products

Genomic DNA of the isolates of interest was amplified using PCR and the fragments of the PCR products were visualised under UV light, using electrophoresis as pictured in Figure 5.

It was shown that the molecular weight of the PCR products was approximately 1.5kb when compared to the molecular weight marker. The intensity of the marker was used to measure the amount required for the 16S rRNA sequencing.Rate of Discovery of New Drugs Assignment Paper

3.6 16S rRNA sequencing result

The 16S rRNA sequences of five isolates of interest were identified and compared using the BLAST tool to determine their species.

Isolates 18 and 71 were classified as Pseudomonas species with isolate 18 having near 100% identity to P. donghuensis. Isolate 71 was identified as P. protegens as both the forward and reverse sequences were 99% identical to the one proposed. On the other hand, isolates 102, 104 and 107 were closely related to Bacillus sp., with the strain unknown. Isolates 102 and 104 were highly similar and both had high possibility to be B. subtilis or B. velezensis as they were 99% identical to these species. For isolate 107, it showed 99-100% similarity with B. pumilus. These results were compatible with the one obtained from gram stain test. Pseudomonas species are Gram-negative and isolates 18 and 71 were shown to be Gram-negative in gram stain, while Bacillus species are Gram-positive, whereby 102, 104 and 107 stained purple in the gram stain test.

Isolate Type Shape

18 G-ve Short rods

33 G-ve Short rods

36 G-ve Coccobacilli

40 G-ve Short rods

71 G-ve Short rods

99 G+ve Rods

101 G+ve Rods

102 G+ve Rods

103 G+ve Rods

104 G+ve Rods

107 G+ve Rods

4. Discussion

Effective antibiotics are essential to maintaining the high standard of healthcare and the emergence of multidrug resistance has urged the process of research and development of novel antibacterial products. The search for new bioactive compounds is necessary to combat these multi-drug resistant pathogens and it might help to delay the progress of antibiotic resistance growth. Aquatic microorganisms have slowly emerged as a new source of active metabolites producing microorganisms and more investigations in this area should be carried out to prompt the success of new discoveries.

In this study, a number of active isolates have been found to exhibit antagonistic activity against a variety of test organisms, showing that there is a synthesis of antimicrobial active compounds in the aquatic life. Five out of eleven isolates were active against at least four test organisms, suggesting the production of wide spectrum antibacterial compounds. The supernatant of the active isolates was tested to determine the antibacterial activity of extracellular metabolites. The overall results from the agar well diffusion showed a generally broader spectrum of activity against the tested bacteria. However, the degree of inhibition was insufficient to make any promising conclusions. Surprisingly, P. aeruginosa was shown to be inhibited by all of the isolates’ supernatant, with a very minimal degree of inhibition. In the 96-well plate test, the OD shown by the samples with E. faecium was the lowest among all, this might be due to its slow-growing property as observed during our study. The increase in OD was detected and it may be caused by the promoting effect on the growth of test organisms or contamination in the samples. The promoting effect shown was significant only for isolates 18 and 107 against P. aeruginosa and this should be further studied. However, the lack of repetition of the OD test may affect the reliability and consistency of the results. Repeated streaking of the active isolates may also lead to the loss of their ability to produce antibiotics or the synthesis of new active compounds due to the variation of growth media (alteration in nutrients, temperature, osmotic conditions etc.)(29). In addition, TSA was used in perpendicular streak test while NB was used for the supernatant test. One of the factors causing limited antibacterial activity shown in the agar well diffusion may be the medium which was possibly less conducive to the production of active compounds.Rate of Discovery of New Drugs Assignment Paper

As shown from the results, the isolated microorganisms showed more activity against Gram-positive bacteria than Gram-negative bacteria. Different sensitivity between the two groups of bacteria has been observed, this could be ascribed to morphological differences of their outer polysaccharide membranes. The outer membrane of Gram-negative bacteria consists of lipopolysaccharide which makes it impermeable to lipophilic solutes whereas the Gram-positive bacteria lack an outer membrane, making them more susceptible and receptive to antibiotics (17). MSSA and MRSA were the most susceptible bacteria to the antibiotics produced by the isolates. This also revealed that the isolates found may be potential to be used to extract the active metabolites that may be useful in treating various severe infections caused by MSSA and MRSA. As expected, P. aeruginosa showed the least susceptibility to the isolates as Pseudomonas species contain innate resistance to a vast array of antibiotics owing to its low outer membrane permeability which acts as a barrier to the uptake of antibiotics and substrate molecules, as well as its active efflux pump system that rapidly pumps the antibiotics out of the bacterial cell (30).

Isolates 18 and 71 were identified as Pseudomonas donghuensis and Pseudomonas protegens respectively. A marine isolate of Pseudomonas sp. has been proven to have the capability of synthesising secondary metabolites that can inhibit a broad spectrum of microorganisms, including K. pneumoniae, MSSA, MRSA, Shigella flexneri, P. aeruginosa and B. subtilis (31). P. donghuensis has demonstrated its ability to excrete a large number of siderophores, including fluorescent pyoverdine and non-fluorescent 7-hydroxytropolones, in numerous studies (32; 33). Under the iron-deficient condition, siderophores act as an iron chelator to increase iron uptake, limiting the amount of iron availability to phytopathogens. It has also demonstrated its antibacterial ability by inhibiting the growth of several virulent species of soft rot plant bacteria from the Dickeya and Pectobacterium genera (33; 34). Due to its iron-chelating properties, P. donghuensis is a potential candidate of biocontrol agent as it can restrict the proliferation and root colonisation by phytopathogens. Isolate 71 was classified as P. protegens, which had been reported that it had plant-protecting ability against a range of soil-borne phytopathogens (35). P. protegens produce secondary metabolites with broad-spectrum antibiotic activity, such as 2,4-diacetylphloroglucinol (DAPG), pyoluteorin, hydrogen cyanide and pyrrolnitrin, where DAPG has been known for its antibiotic and antifungal ability due to its toxicity against a wide range of bacteria, fungi, oomycetes and plants (35; 36). In a study, it was reported that a strain of P. protegens was active against E. coli, S. aureus as well as Bacillus cereus, whereas in another study, P. protegens isolated from treated wastewater had demonstrated multidrug-resistance against ceftazidime, cefepine, ticarcillin-clavulanic acid and aztreonam (37; 38). This shows that P. protegens possess antibacterial activity against numerous plant pathogens, although limited studies have been carried out in human pathogens.

Of all of the isolates, isolate 107 showed the strongest activity, especially against Gram-positive bacteria. Together with isolates 102 and 104, they were classified as part of the genus of Bacillus species. It is not surprising that three microorganisms isolated belong to the genus Bacillus as they have been known as producers of a wide range of biologically active molecules (39). Bacillus sp. are Gram-positive bacteria which are known to generate spores under adverse conditions to ensure their survival in adverse conditions, and both marine and terrestrial isolates of Bacillus have displayed capability of producing potent metabolites (13; 40). Peptide antibiotics produced by Bacillus sp. can be classified into two major groups, (i) non-ribosomally synthesised, such as bacitracin, gramicidin, surfactin, tyrotricidin, and (ii) ribosomally synthesised, such as bacteriocins (12; 41). In the present study, isolates 102 and 104 were identified as either Bacillus subtilis or Bacillus velezensis.

In a few studies, it has been shown that B. subtilis isolates have the ability to synthesise protein antibiotics, like subtilin, subtilosin and sublancin, which are mostly active against Gram-positive bacteria (42-44). This is consistent with our results in the perpendicular streak test where isolates 102 and 104 only inhibited the growth of Gram-positive test organisms. Subtilosin A is one of the antibiotics synthesised by B. subtilis, it has demonstrated to be active against a wide range of bacteria, including Listeria monocytogenes, E. coli, S. aureus, and K. pneumoniae, although its effect on human pathogenic bacteria was moderate (45). Besides antibacterial activity, both B. subtilis and B. velezensis also produce antifungal lipopeptides, such as iturin, bacillomycin, and fengycin, where strains of B. subtilis have demonstrated strong antifungal properties against Aspergillus ochraceus, Penicillium roqueforti, and strains of B. velezensis were active against Alternaria panax and Magnaporthe oryzae. (12; 46; 47).Rate of Discovery of New Drugs Assignment Paper

16S rRNA sequence of isolate 107 has suggested it to be B. pumilus. Limited studies have been conducted using B. pumilus, especially against human pathogens. However, it was found that one strain of B. pumilus produced pumilicin 4, a bacteriocin with good heat and pH stability, and has activity against a vast array of Gram-positive bacteria, including MRSA and VRE (48). In the present study, isolate 104 has demonstrated antibacterial action only against Gram-positive bacteria, which is in contrast to a few studies which have reported that B. pumilus showed inhibition against both Gram-positive (eg: MSSA) and Gram-negative bacteria (eg: E. coli) (49; 50). This shows that B. pumilus could potentially produce more than one type of bioactive compounds (eg: pumilicin, surfactin, iturin) which are active against different classes of bacteria (46; 50). The strain of B. pumilus isolated in our study also showed powerful inhibition against different strains of E. coli. Bacillus species is widely investigated for its effectiveness against pathogenic fungi and plant disease, but only a few studies have been done on human pathogenic bacteria which hinder us from making a constructive conclusion that can contribute to the drug research and development.

In our study, the concentration of the active compounds synthesised by the supernatant was fairly low, which may result in the loss of the antibacterial activity. The culture filtrates can be further purified using solvent extraction procedure in the future to enhance the antibiotic extraction (51). Furthermore, upcoming research should focus on the identification of the bioactive metabolites produced and the extraction of the active compounds synthesised. Scientists should also come up with methods to mass produce the useful products for pharmaceutical purposes. Plenty of research have been conducted on phytopathogens using the isolates that have been found in this study and an emphasis on their effects on human diseases or infections should be discussed in the future. All in all, a number of useful isolates have been found in the present study, showing that the aquatic environment is a potential source to be employed to isolate antibiotic-producing microorganisms.

5. Conclusion

Our findings in the present study have demonstrated that water can harbour microorganisms that are capable of producing antibiotics active against a variety of bacteria, including both Gram-positive and Gram-negative bacteria. Environmental sources apart from soil should be explored for the search of potential bioactive compounds to treat infections, especially those caused by multi-drug resistant pathogens. It is essential to improve on current standard drugs as well as to carry out incessant screening of useful microbial products for the development of new therapeutic agents to counteract drug resistance in the microbial population. In addition, more effective and user-friendly methods of screening and extracting useful metabolites should be invented to ease the process. However, the development of new classes of antibiotics cannot completely prevent the expansion of resistant strains of pathogenic bacteria and hence, antibiotic stewardship and patient education regarding the appropriate use of antibiotics come into play to slow down the progress of the spreading of antibiotic resistance. Rate of Discovery of New Drugs Assignment Paper