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Once diagnosed by gene sequencing, the next step is to design drug to shut off those genes. All three old age diseases, that is, cancer, cardiovascular diseases, and Alzheimer carry multiple mutated genes responsible for causing these diseases. In each of the above three diseases, it is the mutated genes that code for wrong protein which causes these diseases.

If we design drugs to shut off mutated genes in one disease, using the same rationale, we should be able to shut off bad genes in all three old age diseases.

Although coronary artery disease is a complex disease, researchers have found about 60 genomic variants that are present more frequently in people with coronary artery disease.

Most of these variants are dispersed

across

the genome and do not cluster on one specific chromosome. Drugs are designed to seek out the specific malignant gene, which replicates faster producing acids. Aziridines and carbamate moieties are sensitive to acid.

Drugs carrying the aziridines and carbamate moieties are broken down in acidic media generating carbonium ions which attack DNA

shutting

off genes. Only the acid producing genes will be attacked no matter where they are located.

It does not matter whether they are clustered or dispersed across genome. The supreme intellect for drug design is Ross, an Englishman, who is a Professor of Chemistry at the London University. Professor WCJ Ross is also the Head of Chemistry Department at the Royal Cancer Hospital, a postgraduate medical center of the London University. Ross was the first person who designed drugs for treating cancers. He designed drugs to cross-link both strands of DNA that we inherit one strand from each parent. Cross-linking agents such as nitrogen mustard are extremely toxic and were used as chemical weapon during the First World War.

More toxic derivatives were developed during the Second World War.

Using the data for the toxic effect of nitrogen mustard used during the First World War, Ross observed that soldiers exposed to nitrogen mustard showed a sharp decline of white blood cells (WBC) that is from 5000 cell/CC to 500 cells/CC. Children suffering from childhood leukemia have a very high WBC count over 90,000 cells/CC.

In sick children, most of the WBCs are premature, defected, and unable to defend the body from microbial infections. Ross rationale was that cancer cells divide faster than the normal cell, by using nitrogen mustard to cross-link both strands of DNA, one can control and stop the abnormal WBC cell division in leukemia patients.

Professor Ross was the first person to synthesize a large number of derivatives of nitrogen mustard. By using an analog of nitrogen mustard, called chlorambucil [9], he was successful in treating childhood leukemia. In America, two physicians named Goodman and Gilman from the Yale University were the first to

use

nitrogen mustard to treat cancer in humans. Nitrogen mustards and its analogs are highly toxic.

Ross was a chemist; over the years, he synthesized several hundred

derivatives

of nitrogen mustard molecules to modify toxicity of nitrogen mustard [10, 11, 12, 13, 14]. Although analogs of nitrogen mustard are highly toxic, they are more

toxic

to cancer cells and more cancer cells are destroyed than the normal cells.

Toxicity is measured as the chemotherapeutic index (CI), which is a ratio between toxicity to cancer cells versus the toxicity to normal cells.

Higher CI means that the drugs are more toxic to cancer cell. Most cross-linking nitrogen mustard have a CI of 10, that is, they are 10 times more toxic to cancer cells.

Some of the nitrogen mustard analogs Ross made over the years are useful for treating cancers such as chlorambucil for treating childhood leukemia (which brought down

the

WBC level down to 5000/CC).

Childhood leukemia is the name of a disease occurs in children only. Chlorambucil made Ross one of the leaders of the scientific world. He also made melphalan and myrophine for treating pharyngeal carcinomas [15]. 6.2 The discovery of AZQ (US Patent 4,146,622) for treating brain cancer.

At the London University, I was trained as an organic chemist in the Laboratory of Professor WCJ Ross of the Royal Cancer Hospital, a postgraduate medical center of the London University. After working for about 10 years at the London University, I moved to America when I was honored by the Fogarty International Fellowship Award by the National Institutes of Health, NIH, and the National Cancer Institute, NCI, of the USA. NIH has been my home for over a quarter of a century; I designed drugs to shut off mutated genes. All three common allele diseases have genetic origin. The rationale I used to synthesize anticancer drugs could be used to treat the other two old age diseases like Alzheimer and cardiovascular diseases.

In the following sections, I will describe in detail how anticancer drug like

AZQ

was designed to shut off glioblastoma genes which cause brain cancer in humans. Using the

same

rational, we will consider how each of the other two

diseases

, namely, cardiovascular disease and Alzheimer could be treated by shutting off their genes to save human life: The order of these diseases are arranged based on the level of funding provided by NIH specifically by the NCI (National Cancer Institute).

As I said above, Professor Ross was designing drugs to attack both strands of DNA simultaneously by cross-linking using nitrogen mustard analogs, which are extremely toxic.

As a part of my doctoral thesis, I was assigned a different path.

Instead of cross-linking DNA, I am to design drugs to attack only one strand of DNA.

Over the years, I made over 100 dinitrophenyl aziridine derivatives.

One of them is dinitrobenzamide (CB1954) which gives a CI of 70, highest ever recorded. CB1954 wipes out a solid tumor by attacking the DNA of Walker carcinoma 256, a solid aggressive tumor in rat.

Nitrogen mustards are highly toxic because they have neither specificity nor selectivity. They attack all dividing cells whether they are normal or abnormal. On the other hand, the analogs of aziridines and carbamates remain inactive in the basic and neutral media. They become activated only in the presence of acidic media. I used a simple rationale, the aziridine attacks DNA in acidic medium, particularly the N-7 guanine.

The dye dinitrobenzamide has great affinity for Walker tumor [16, 17, 18].

The aziridine dinitrobenzamide (CB1954) stains the tumor.

As the tumor grows, it uses glucose as a source of energy. The ring opens to generate a carbonium ion, which attacks the most negatively charged N-7 guanine of DNA shutting off the Walker carcinoma gene in rat. To continue my work, I was honored with the Institute of Cancer Research Post-Doctoral Fellowship Award of the Royal Cancer Hospital of London University.

To increase the toxicity of CB1954 to Walker carcinoma, I made additional 20 analogs as a postdoctoral fellow.

When I attached one more carbonium generating moiety, the carbamate moiety to the aziridine dinitrobenzene, the compound aziridine dinitrobenzamide carbamate was so toxic that its therapeutic index could not be measured.

We stopped the work at the London University for the safety concern. I continued my work on the highly toxic aziridine/carbamate combination in America when I was offered the Fogarty International Fellowship Award to continue my work at the National Cancer Institute (NCI) of the National Institutes of Health (NIH).

I brought the idea from London University of attacking one strand of DNA using not only aziridine, but also carbamate without using the same dye dinitrobenzamide [19, 20, 21]. My greatest challenge at NCI is to translate the animal work which I did in London University to humans.

One day, I came across a paper which described that radiolabeled methylated quinone cross the blood-brain barrier in mice. When injected in mice, the X-ray photograph showed that the entire radioactivity was concentrated in the mice’s brain within 24 hours. I immediately realized that glioblastoma multiforme, the brain

tumor

in humans, is a

solid

aggressive tumor like Walker carcinoma in rats.

I decided to use quinone moiety as a carrier for aziridine rings to attack glioblastomas.

By introducing an additional carbamate moiety, I could increase its toxicity several folds.

I planned to use this rational to translate animal work to human by introducing multiple aziridine and carbamate moieties to the quinone to test against glioblastomas in humans.

Attaching two aziridines and two carbamate moieties to quinone, I made AZQ. By

treating

brain cancer with AZQ, we observed that glioblastoma tumor not only stop growing but also start shrinking.

I could take care of at least one form of deadliest old age cancers, that is, glioblastomas. Literature search showed that AZQ is extensively studied. As I said above, glioblastoma, the brain cancers, is a solid and aggressive tumor and is caused by mutations on several chromosomal DNA. Mutations on DNA are the result of damaging DNA nucleotides by exposure to radiations, chemical and environmental pollution, viral infections, or genetic inheritance.

The other factors responsible for causing DNA mutations are due to the fast rate of replication of DNA. coli grows so rapidly that within 24 hours, a single cell on a petri dish forms an entire colony of millions when incubated on the agar gel. Rapid replication is responsible for introducing genetic defects causing diseases. When an additional piece of nucleotide is attached to a DNA string, it is called insertion or a piece of DNA is removed from the DNA string; it is called deletion or structural inversion of DNA is responsible for mutations. Since the gene in a DNA codes for proteins, insertion and deletion on DNA have

catastrophic

effects on protein synthesis. In glioblastomas, three major changes occur on chromosomes (C-7, C-9, and C-10) and two minor changes occur on chromosomes (C-1 and C-19). These mutations are responsible for causing brain cancers in humans.

In a normal human cell, chromosome-7 which is made of 171 million nucleotide base pairs and carries 1378 genes.

When insertion occurs on chromosome-7, 97% of glioblastoma patients are affected by this mutation.

On the other hand, a different mutation occurs on chromosome-9 which is made of 145 million nucleotide base pairs and it carries 1076 genes. A major deletion of a piece of DNA occurs on chromosome-9, which results in 83% patients who are affected by this mutation.

A minor deletion of DNA also occurs on chromosome-10 which is made of 144 million base pairs and it carries 923 genes.

Although it is a minor deletion of a piece of DNA, it contributes to 91% patients with glioblastoma.

To a lesser extent, small mutation occurs on chromosome-1 (the largest chromosome in our genome).

It is made of 263 million nucleotide base pairs and carries 2610 genes), and chromosome-19 (it is made of 67 million base pairs and carries 1592 genes) is also implicated in some forms of glioblastomas. All known glioblastomas causing genes are located on five different chromosomes and carries a total of 9579 genes.

It appears impossible to design drugs to treat glioblastomas since we do not know which nucleotide on which gene and on which chromosome is responsible for causing the disease.

With the completion of 1000 Human Genome Project, it becomes easier.

By simply comparing the patient’s chromosomes with the 1000 genomes, letter by letter, word by word, and sentence by sentence, we could identify the difference called the variants with precision and accuracy, the exact variants or mutations responsible for causing the disease.

Once the diagnosis is confirmed, the next step is how to treat the disease.

With the quinone ring, I could introduce different combinations of aziridine rings and carbamate moieties and could create havoc for glioblastomas. My major concern was how toxic this compound would be to the human brain cells. Fortunately, brain cells do not divide, only cancer cells divide. Our rational drug design work began in the University of London, England, and completed in the Laboratory of the National Cancer Institute (NCI), of the National Institutes of Health (NIH), in Bethesda, Maryland, USA.

Over this period, we conducted over 500 experiments which resulted in 200 novel drugs.

They were all tested against the experimental animal tumors.

Forty-five of them were considered valuable enough to be patented by the US Government (US Patent 4,146,622).

Radiolabeled studies showed that AZQ has the ability to cross organ after organ, cross the blood-brain barrier, cross the nuclear membrane, and attack the nuclear DNA shutting off the gene.

X-ray studies showed that the radioactivity is concentrated in the tumor region. For the discovery of AZQ, I was honored with the “2004 NIH Scientific Achievement Award,” one of America’s highest awards in Medicine and I was also honored with the India’s National Medal of Honor, “Vaidya Ratna,” a gold medal (see Figures 1–4). 2004 NIH Scientific Achievement Award presented to Dr. Elias Zerhouni, the director of NIH

during

the NIH/APAO award ceremony held on December 3, 2004. Khan ww951 white is the discoverer of AZQ (US Patent 4,146,622), a novel experimental drug specifically designed to shut off a gene that causes brain cancer for which he receives a 17-year royalty for his invention (license number L-0I9-0I/0).

To this date, more than 300 research papers have been published on AZQ.

The award ceremony was broadcast live worldwide by the Voice of America (VOA).



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