keegan asked:


And now for the perhaps shocking news: there are times when Viagra, Cialis and Levitra are of no help at all for treating erectile dysfunction. You have all been persuaded to believe that Viagra and the other PDE-5 inhibitors will always be able to help, even if only a little. Unfortunately, that’s not true. There are times when the degree of damage is too great for a “natural” solution. And let’s be clear. In relative terms, the use of Viagra and the others allows the body to respond to sexual stimulation as it was designed to do. Viagra simply removes a physical barrier to a natural ********.

This means we are into the surgical options. The first to mention is vascular reconstructive surgery. In a small number of cases, the surgeon may be able to reconstruct the arteries so that blood will flow more freely into the corpus cavernosum. Alternatively, surgery may help those suffering from venous leakages. When the blood inflates the corpus cavernosum, it compresses the veins that allow the blood to drain out of the *****. If the compression is inadequate, the blood will not be retained inside the corpus and the ******** will deflate. Unfortunately, the surgical theories have mostly failed in practice. Only about 5% of those having reconstructive surgery have found improved performance. This surgery is expensive. It is a long and difficult operation, and any incision can cause nerve damage. So it is not often used today.

Thus, when all else has failed or is not acceptable for some reason, we come to the somewhat radical solution of implanting a penile prosthesis.

How does this work?

There are three tubes in the penile shaft. Running along the bottom of the penis, the urethra drains the urine from the bladder out of the body through the *****. Along the top of the penis, the two more substantial tubes run side-by-side. They are designed to work in much the same way as the tyre on a car, i.e. with an inner and outer tube. So when the inner tubes fill with blood, they push against the outer tubes and, as the volume of blood increases, lift the ***** into an erect position.

Since the 1930s, medical researchers have been experimenting with implants to mimic this process of inflation and create the effect of an ********. We therefore have more than seventy years of experience among more than one million men to draw on. Two different approaches have come to dominate. One range of implants remains semi-rigid. These devices are easy to implant and very rarely fail mechanically. The problem, however, is that they produce a permanent ******** which can be difficult to conceal. Although they may be flexed up and down, most have limited mobility. The Dura II manufactured by American Medical Systems (AMS) has a more sophisticated system relying on interlocking disks strung on a central cable under tension which offers greater control over positioning, but may have an increased risk of mechanical failure.

The others are hydraulic, e.g. Dynaflex and CXM, and inflate, relying on a pump contained inside the ******* to transfer fluid (almost always water) from a reservoir inside the abdomen. These devices may have:

1.one, rod-like inflatable chamber which is hinged;

2.a two chamber system, e.g. the Ambicor; or

3.a three chamber system. All these systems replace the original corpa cavernosa. Today’s three-piece inflatable devices produced by AMS and Mentor Corporation offer flaccidity and ********* that approach the natural state. Whichever system you opt for, you should expect some pain and discomfort for five or six weeks following surgery. And, once installed, the implants cannot replace the “real thing”. Thus, if you have already lost your libido, it will not return. Similarly, if the nerve damage had caused a loss of sensation, or you were unable to achieve ****** or ejaculation, prosthetics will not restore those functions.So what are the advantages of this technology? Well, when the oral medications such as Viagra, Cialis and Levitra have failed, the convenience factors of the alternatives become important. Implants are safe and work no matter what the original cause of the ED. They are particularly useful to treat cases of damage following priapism and Peyronie’s Disease, fibrosis after injections, and to restore function after surgery for prostate cancer. So long as the partners are counselled on the strengths and weaknesses of the systems available, they provide satisfaction in more than 90% of all cases. Once installed, there are no further costs and they require no further maintenance (only about 3% of all installed devices ever need attention). From a psychological point of view, there is no external equipment to distract — as in the case of injections and vacuum devices. Equally, there is no need to remember to bring the Viagra, Cialis or Levitra along. If you are in the mood, you can simply act without having to wait for the medication to take effect.

Nevertheless, there are disadvantages to consider. This is a once-and-for-all procedure. Once the implant has been installed, it is no longer possible for a natural ******** to occur. It is always possible that, as science develops, different non-invasive procedures may become available. One may solve the ED problem you have. If your view is that a more natural form of treatment is better, then you may decide to wait. On the issue of informed consent, there are always risks in surgery involving an anaesthetic. In particular and despite a coating of antibiotics to modern implants, you will learn there is a small risk of infection following the surgery. This is relatively rare, but if it does occur and irrigation fails, the only response is to remove the prosthesis. However, once installed, there are three more general complaints.

1.Because the ******** is not being caused “naturally”, the systems only produce hardness in the body of the *****. Neither system affects the head of the ***** which remains soft. This can feel unsatisfactory and make *********** uncomfortable.

2.The body of the ***** does not warm as in the natural ******** which can feel strange to your partner.

3.The surgery can sometimes produce a shorter ***** which you and your partner may find unsatisfactory.

Finally, let’s dispose of one more general worry that some of you may have. There have been a number of problems with the safety of some silicone implants, e.g. in ****** enhancements. None of the inflatable prostheses use any form of silicone as the fluid.

Niels Grønning Rasmussen asked:


Preface

 

A growing industrial demand for high throughput screening of new molecular entities (NME) has spurred great advances in various divisions of scientific development. Researchers has begun to combine various scientific methods to meet the competitive demands in modern drug development, and as a consequence optimized individual phases of drug development.

 

Technological improvements should ensure high quality expeditious drug development and a as a consequence a modern “fast track” from drug discovery to marketed product.

 

These scientific advances should however be viewed in sharp contrast to the increased economical expenditures observed in pharmaceutical companies worldwide. The price from discovery phase to actual marketing is estimated to be in the region of 0.8 billion to 1.7 billion dollars (11) and research has shown that investments in NME’s has increased 55 % over the last five year (12). These economical prospects are paradoxically followed by the fact that there has been observed a mere 7% increase in FDA-approved NME’s from 1993 to 2003. These combined factors are inherently linked to a new paradigm shift in drug development where technological advances outrun the collaborative marketing of new drugs.

 

This project will present the reader with three established analytical methods in drug development and link these methods with nine analytical properties proposed by M. Valcárel and A. Rios (2). Implementation of these methods in drug development and individual quality standards and guidelines will be presented and discussed and the project will be summarised with concluding remarks.

 

The nine analytical properties

The analytical properties proposed by M. Valcárel and A. Rios (2) should be regarded as a framework which efficiently introduces the students to the various aspects of analytical chemistry. They are a means of providing a hierarchical approach toward the analytical chemistry curriculum and should ensure an organized introduction to analytical chemistry.

 

The significance of introducing analytical properties hierarchically to students can be described in terms of the following three groups proposed by the authors. The most important group is the top or capital group. This group is defined by the need for accurate and representative data which in turn is the sole contributor towards the quality of the results.

 

The second group, the basic properties, ensure the quality of the analytical process and cover the more practical aspects of analytical chemistry. They include such properties as selectivity, precision, sensitivity and proper sampling. They are properties which during the course of an analytical approach can prove contradictory and could lead to a great deal of decision making in the process. The decisions which are made could alter the quality of the analytical processes and in turn influence the quality of the results. The scientist must reflect on these basic properties and establish a standard by which the analytical processes are conducted in order to facilitate high quality analytical processes. The various analytical factors their complementary and contradictory relationships (2) fig. 4) should be evaluated and the analytical research should be conducted accordingly.

 

The quality of the analytical process and the quality of the results are thus highly dependent on one another, and it should become apparent that the capital properties and the basic properties have a mutual relationship in obtaining high quality results meeting external quality standards. The accuracy and representativeness defined by the capital properties is only relevant if we ensure strict precision, sensitivity, selectivity and proper sampling in the laboratory.

 

The last group of properties, the accessory properties, are interesting in respects to their mutual influence and relationship with the basic properties. The accessory properties are represented by expeditiousness, cost-effectiveness and personnel safety and comfort. These are properties reflecting the competitive aspects of analytical chemistry and are mutually dependent on the basic properties. Thus, compromises regarding sensitivity, precision and selectivity could yield greater expeditiousness and improved productivity compared to competing companies or laboratories. There are however contradictory aspects of the accessory properties and reducing the basic properties could potentially influence various accessory properties thus leading to further challenges in the productivity and competitiveness.

 

The properties proposed by M. Valcárel and A. Rios should be thought of as a way to hierarchically introduce analytical chemistry. Each group represents a significant part of analytical chemistry and should be dealt with thoroughly by the teacher in order to facilitate a correct understanding by the individual student. The individual groups thus represent the framework by which we can introduce the student to analytical chemistry and rightfully cover the curriculum. It also reflects contradictory relationships in analytical chemistry and proposes some interesting aspects concerning competitiveness, expeditiousness and sensitivity in the laboratory. In industrial context the properties illustrate the paradox observed then assessing various pharmaceutical projects and they are an excellent way to graphically present various dilemmas in drug development.

 

Analytical techniques in drug design and discovery

Current initiatives in analytical chemistry propose some interesting aspects by combining various division of science towards high yield quality data. Some of these methods are successfully implemented in various phases of drug development and aid in shortening the timeline from lead compound to marketed drug. A collection of these methods are presented below and they should present the reader with a quick overview on some industrially applied analytical techniques. These methods equally show promising results towards scientific advances in analytical chemistry and they demonstrate the implementation of “lean” strategic guidelines in modern drug development and discovery.

 

“Development of a Ubiquitin Transfer Assay for High Throughput Screening by Fluorescence Resonance Energy Transfer” (3).

 

Boiscalir et. al (3) propose a new assay based on fluorescence resonance energy transfer (FRET) which successfully could lead to high throughput real time screening of the ubiquitination process in vitro. They furthermore challenge the novel DELFIA assay and suggest that the FRET assay shows lower data variability and due the simplicity of the FRET analysis, the tedious washing, binding and incubation steps in DELFIA are avoided.

 

Protein degradation by means of ubiquitination involves three enzymatic steps (E1 to E3). Each of these enzymes contributes to the ubiquitination process which leads to the covalent attachment of ubiquitin with ?-aminoacid lysine residues on the target protein (6). The binding of ubiquitin targets the protein for degradation by the proteasomes.

 

The suggested assay measures the transfer of ubiquitin between the E2 enzyme (Ubc4) and Rsc (human HECT protein). By coupling the Rsc protein to glutathione-S-transferase (GST) and using an anti-GST antibody labelled with Eu3+ it is possible to pre-label the Rsc-GST fusion protein.

 

The assay exploits the fact that the upon excitation of the compounds, the flourophores Eu3+ and APC exhibits energy transfer and the emission experienced is directly measurable at 665 nm (APC) and 615 nm (Eu3+). To introduce the APC flourophore, the authors have created an ubiquitin-biotin-Streptavidin-Apc complex. Eu3+ acts as the energy donor while APC acts as the energy acceptor, when these are in close proximity (less then 100 Å) to one another, emission is observed from Eu3+ at 615 nm and at 665 nm with APC. The signal intensity presents a means to quantify the ubiquitination process real-time.

 

The results presented in the paper and the sophisticated experimental set-up revels promising real time data acquisition of biological processes and the method could become a valuable tool in either lead optimization or could successfully be implemented in future HTS assays.

 

“Drug screening of pharmaceutical discovery compounds by micro-size exclusion chromatography/mass spectrometry” (4).

 

Paul A. Wabnits and Joseph A. Loo propose a new method which successfully could improve the screening of active ligands in molecular binding studies. Micro-size exclusion chromatography offers a fast and simple way to separate the free ligand from the bound protein-ligand complex. Coupled with liquid chromatography and mass spectrometry the authors present a quick and efficient assay which potentially could lead to high throughput molecular binding quantification.

 

The metalloenzyme peptide deformylase (PDF) was chosen as the target protein, but in order to achieve sufficient spectroscopic data, the authors exchanged the native Fe2+ with the spectroscopically active Co2+. The binding assay was performed on PDF with several suspected inhibitory drugs (the complete structure was only revealed for actinonin due to commercial reasons).

 

Various ligands were incubated with PDF, and by means of micro-size exclusion chromatography and mass spectrometry, it was possible to obtain mass spectrums revealing the presence or absence of free ligands. The presence of free ligand should indicate weak/no binding and by performing this with various combinations of drugs the researchers where able to establish the binding rank order of the drugs ((4) table 3).

 

These data suggest the rank by which various drugs show inhibition of the PDF enzyme, and furthermore introduces a method which could yield fast screening of therapeutic leads towards potential macromolecular targets. The procedure shows high sensitivity and enables researcher to perform fast efficient binding studies of various protein and ligand combinations.

 

The proposed micro-size exclusion principle is furthermore a great example of the simplicity observed in recent advances in drug development and discovery.  

 

“In vitro identification using fast gradient high performance liquid chromatography combined with tandem mass spectrometry” (5).

 

In order to perform quick efficient separation of novel drugs and their metabolites, a growing need for optimization of already established analytical techniques is required. A reduction in the time by which researchers can assess potential drugs and their metabolites could be proven valuable in the lead optimization stage of drug design. Even a small reduction in the identification time is of great interest economically and professionally, and could lead to an enhancement in this important phase of drug design.

 

Coupling fast gradient high liquid chromatography with tandem mass spectrometry could reduce the bottleneck observed in the lead optimization stage as proposed by Cornelis et. al.

 

The authors present a fast and efficient way to separate and identify isomeric drug metabolites as well as the parent compound, by use of already established analytical processes. Microsomal incubation of the novel drugs produces the suspected metabolites which by means of fast gradient high liquid chromatography are successfully separated. The separation process is proven by coupling the chromatographic process with tandem mass spectrometry. This enables the researcher to establish the presence or absence of various metabolites and indicates the metabolic fate of the investigated drug.

 

By reducing analysis time researchers could reduce this step and Cornelis et. al propose a technique which successfully could separate metabolites from parent compound in less than 2 minutes. Optimal conditions were obtained upon increasing the flow rate to 2000 µL/min, and enabled successful chromatographic separation of the metabolites and the pure compound ((6) fig. 2+3).

 

The method is thus of industrial significance due to the time reduction observed in the process and the relatively easy experimental set-up.

 

 

Lead discovery and optimization

 

High throughput screening (HTS)

Industrial high throughput screening (HTS) presents a multidisciplinary science protruding various scientific fields of study. HTS has become a standard in modern drug development signified by a new paradigm shift toward automatization in the lead discovery phase. It is no longer unusual, that pharmaceutical companies have huge screening libraries containing between 500.000 and 1.000.000 compounds (8), controlled by robotics and computational devices. These libraries provide an extensive database containing valuable compound information which linked to recombinant “humanized” in vitro tests could improve expeditiousness and thus as a consequence improve the drug development and discovery process. The libraries should however not be perceived as a clear cut improvement in the lead discovery phase. The expeditiousness of a lead finding is highly dependent on the development of independent assays modelled towards the suspected target. This could potentially produce a bottleneck in discovery phase and should thoroughly be debated in project decision making.

 

 Criteria’s in HTS

The dawn of these enormous screening libraries has sequestered a demand for assays which are able to successfully screen thousands of compounds and furthermore provide sustainable data fast and efficient. This has forced researchers to optimize existing analytical techniques and implement various branches of natural science towards the creation of assays which fulfil specific standards.

 

The ideal HTS assay should exhibit the following properties (8):

High signal to background ratio (S/B) or signal to noise (S/N) High z´-factor (9) (values >0,5 represent a good assay) Fast screening time Low coefficient of variation (CV %) Large Stokes Shift



These properties are quantifiable measures aiding the researcher in producing high quality HTS assays modelled towards the proposed target. These criteria thus impose boundaries and guidelines which if followed properly optimistically results in ideal screening methods for future implementation in the drug development phases. Even the smallest improvement in one of the parameters could result in higher expeditiousness and potentially a shortening of the individual developmental phases.

 

As stated above, efficient screening of compounds is essential in locating the perfect lead candidate for future studies. Observing the parameters, it is evident that there are colliding interests between various aspects of analytical chemistry and economical interests. HTS could be grouped with the above mentioned accessory properties in the nine analytical properties (1,2), reflecting upon the balance between expeditiousness and cost effectiveness in terms of economical profitability. It stands in sharp contrast to the basic properties ensuring high precision, sensitivity and selectivity.

 

There is a fine balance between producing high throughput data and obtaining the adequate precision needed to produce quality results which live up to internal/external standards. As a consequence, the decisions we choose in these two groups highly reflect the accuracy and representativeness found in the capital properties and thus the quality of the results. These conflicting properties should be evaluated in the early drug development phases and it is the research and development team’s job in collaboration with the financial team to define internal standards for which they both can agree upon.

 

Implementation of quantitative techniques in drug development

 

HTS assays implemented in the early discovery phase are a powerful tool in establishing the lead compounds interaction with a suspected biological target. Once a suitable lead has been found quantitative studies are needed to further elucidate the leads biological and chemical characteristics. Quantitative studies following these screening assays provide direct quantifiable data concerning the leads metabolic, pharmacokinetic and toxicological fate. These studies provide valuable information concerning the leads biological properties, and the data obtained in the quantitative studies informs the researchers about potential problems such as toxicology, absorption, distribution excretion, pharmacodynamics and metabolism. Quantitative studies are thus often observed in lead optimization studies where they provide important experimental data.  

 

These studies rely on analytical methods with great precision, expeditiousness, sensitivity and they inherit some of the same criteria as HTS assays (se above). Deviations to the above mentioned criteria’s exist and they include the following:

The need for an adequate internal standard Heightened Precision (of great importance in e.g. toxicological studies) High Selectivity Low matrix interference



When conducting these quantitative studies it is of great importance that the data obtained reflects the precise action of the drug and that the methods inherently refrains from producing false positives. It is obvious that quantification of a seemingly unknown drug is difficult. Purification and the construction of an internal standard is alone a tedious task which requires a great deal of experimental work. Introduction of the lead drug into the lead optimization stage furthermore requires the development of specific modelled analytical techniques which potentially could resolve in a bottleneck in the specific phase.

 

As stated above there is a clear cut difference between the quantitative studies and the screening methods employed upon implementing assays and procedures in drug design and development. Screening has obvious advantages in the lead discovery phase, and is a strong tool which efficiently and swiftly screens numerous compounds for desired characteristics. The screening assays are however restricted upon relying on a great deal of compound information concerning the proposed target and lead compounds. Structural information concerning pKa, lipphilicity, LogP and LogD is needed to develop an assay that follows the above mentioned success criteria. However, once developed, a successful assay generates valuable data for further use and is imperative when evaluating potential lead candidates in the lead discovery phase.   

 

The quantitative study presented by Cornelis et. al (5) is an excellent example of a procedure which could be deployed in the lead optimization stage of drug development. The metabolic fate of the lead compound can be evaluated and quantification is possible in specific ADME/Tox studies. However, the procedure is dependent on extensive knowledge of the mass and structure of the metabolite or drug compound in order to adequately ascertain that we quantify the correct entity.

 

In conclusion

 

The drug development and discovery phases introduce some interesting possibilities for various implantation strategies in respects to scientific collaborations and technological advances. One recent advance in liquid chromatography is the dawn of ultra performance liquid chromatography (UPLC) which shows promising results toward quantitative studies (14). The current trends thus reveal great potential in implementation of these new analytical techniques and future studies may contribute to heightened quality in the lead discovery and optimization phases.

 

The increased governmental requirements for quantitative ADME/Tox studies has forced pharmaceutical companies to invest enormous amounts of money in order to fulfil requirements suggested by the FDA or other local medicines agencies. This has prolonged the development from lead compound to marketed drug and I suggest that this indeed has faltered a paradigm shift in modern drug development. New biotech companies and scientific entrepreneurs are now faced with tough decisions regarding financial aspects in order to ***** the sustainability of a suspected leads and they should rightfully evaluate the various properties presented by M. Valcárel and A. Rios. We are positioned in a technological vacuum where technological advances greatly exceed the introduction of new drugs towards patients and the industrial drug development is faced with exciting challenges for the future which successfully may increase the amount of drugs marketed per year.

 

References 

1. Valcárcel M. A modern definition of analytical chemistry. Trends in Analytical Chemistry. 1997. 16. pp 124-131 

2. Varcárcel M and Rios A. Teaching analytical properties. Fresenius Journal of Analytical Chemistry. 1997. 354. 202-205. 

3. Michael D. Boisclair, Christopher McClure, Serene Josiah, Susan Glass, Steve Bottomley, Shubi Kamerkar and Ilkka Hemmilä. Development of a Ubiquitin Transfer Assay for High Throughput Screening by Fluorescence Resonance Energy Transfer. Journal of Biomolecular Screening, Vol. 5, 2000, pp. 328. 

4. Paul A. Wabnitz and Joseph A. Loo. Drug Screening of pharmaceutical discovery compounds by micro-size exclusion chromatography/mass spectrometry. Rapid communications in mass spectrometry, Vol. 16, 2002, pp. 85-91. 

5. Cornelis E. C. A. Hop, Phillip R. Tiller and Leslie Romanyshyn. In vitro metabolite identification using fast gradient high performance liquid chromatography combined with tandem mass spectrometry. Rapid communications in mass spectrometry, Vol. 16, 2002, pp. 212-219. 

6. Jeremy M. Berg, John L. Tymoczko, Lubert Stryer. Biochemistry 5th edition. W. H. Freeman and Company. 

7. György M. Keser? and Gergely M. Makara. Hit discovery and hit-to-lead approaches. Drug Discovery Today. Volume 5, Issue 7, 1 July 2000, Pages 286-293.

8. H P Rang. Drug Discovery and Development. Churchill Livingstone, Elsevier. 2006. 

10. Alfonso Espada, Manuel Molina-Martin, Jeffrey Dage2 and Ming-Shang Kuo. Application of LC/MS and related techniques to high-throughput drug discovery. Drug Discovery Today. Volume 13, issue 9-10, May 2008, Page 417-423.

11. Tufts Center for the Study of Drug Development, Backgrounder: How New Drugs Move Through the Development and Approval Process, Boston: November 2001

12. Windhover’s In Vivo: The Business of Medicine Report, Bain drug economics model, 2003 

13.